{"paper_id":"cd0131da-613a-47c3-be38-e1f2ed231da6","body_text":"1 \nAn elevated environmental temperature impairs accumulation of the pattern \nrecognition receptor FLS2 \nShort title: Heat reduces FLS2 accumulation \nBryony C.I.C. Jacobs1†*, Kyle W. Bender2, Emma Six2, Cyril Zipfel2,3, and Marc R. \nKnight1* \n1Department of Biosciences, Durham University, South Road, Durham DH1 3LE, UK. \n2Department of Plant and Microbial Biology, Zurich-Basel Plant Science Center, \nUniversity of Zurich, 8008 Zurich, Switzerland. \n3The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, Norwich \nNR4 7UH, UK. \n†Current address: Department of Plant Sciences, University of Cambridge, Downing \nStreet, Cambridge CB2 3EA, UK. \n \nJoint authors for correspondence: Marc R. Knight \n(m.r.knight@durham.ac.uk) and Bryony C.I.C Jacobs (bcj27@cam.ac.uk).  \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 2 \nABSTRACT \nPattern-triggered immunity (PTI) is initiated when plants detect pathogen -associated \nmolecular patterns (PAMPs)  through patter n-recognition receptors (PRRs).  How \nmoderate increases in temperature affect this plant immune signalling remains unclear. \nWe explored this by using flg22  and the leucine -rich repeat receptor kinase (LRR -RK) \nFLS2 as a model receptor -ligand system and Ca 2+ signaling as a representative PTI \noutput. A pre-treatment at 28  °C significantly impair ed the flg22-induced [Ca2+]cyt influx, \nleading to a reduced expression of calcium-dependent defence genes, ICS1 and EDS1. \nThis effect correlated with a temperature -dependent reduction in FLS2  abundance. A \nqualitatively similar inhibition of these responses was observed when membrane fluidity \nwas artificially increased using benzyl alcohol.  This suggests that the effect of elevated \ntemperature might act through changes in membrane properties. Artificially restoring \nFLS2 protein levels rescue d flg22-dependent Ca2+ signalling and ICS1 and EDS1 \nexpression in seedlings pre -treated at 28°C or with benzyl alcohol. Together, these \nfindings indicate that increased membrane fluidity reduces FLS2 protein levels , thereby \ncompromising Ca2+ signalling, and probably other, flg22-indcued responses . This \nhighlights a potential mechanistic link between temperature perception, memb rane \nfluidity, and FLS2-dependent calcium signalling, providing insight into how an increase in \nglobal temperatures may compromise plant immune responses in the future. \n \nINTRODUCTION \nPlants are continually challenged by fluctuating, and often devastating , environmental \nconditions. To optimise growth and fitness, they must rapidly and precisely assess \nconditions to activate an appropriate change in internal physiological processes. Central \nto this ability are complex cell signalling networks that detect and relay environmental \ninformation and coordinate an informed integrated response in context with other factors \nsuch as biotic stresses.  \nThe second messenger calcium is a key orchestrator of responses to environmental \nparameters, converting external stimuli into intracellular signals that can activate the \nappropriate downstream responses. The properties of cytosolic calcium responses have \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 3 \nbeen found to be stimulus -specific (McAinsh & Hetherington, 1998) . These signals \npossess characteristic  and specific  kinetics and so are termed “calcium signatures” \n(McAinsh & Pittman, 2009) . Emerging evidence suggests these calcium signatures are \ndecoded by calcium -binding proteins, including calmodulin, calmodulin -like proteins, \ncalcium-dependent protein kinases and calcineurin B -like proteins , to effect specific \nappropriate downstream responses  (Hashimoto & Kudla, 2011; Poovaiah & Du, 2018; \nKudla et al., 2018). Despite this knowledge, a n intriguing question remains: do different \ncombinations of environmental factors modify a calcium signature to make it contextually \nappropriate? \nIn the context of plant defence against microbial pathogens, calcium signalling plays a \npivotal role in patten -triggered immunity (PTI), which is initiated by the recognition of \npathogen-associated molecular patterns (PAMPs) by plant pattern recognition receptors \n(PRRs) (Jones & Dangl, 2006) . A paradigm example is the perception of the bacterial \nflagellin-derived peptide , flg22, by the plasma -membrane leucine-rich repeat receptor \nkinase FLAGELLIN SENSING 2  (FLS2) (Felix et al. , 1999; Gómez -Gómez & Boller, \n2000). The binding of flg22 to FLS2 causes a rapid increase in cytosolic calcium \nconcentration [Ca 2+]cyt, mostly through the influx of calcium from external stores  \n(Jeworutzki et al., 2010). This elevated calcium signal initiates, amongst other events, the \nexpression of defence genes including ISOCHORISMATE SYNTHASE  1 (ICS1) and \nENHANCED DISEASE SUSCEPTIBILITY  1 (EDS1), which mediate salicylic acid -\ndependent immune responses  (Lenzoni et al., 2018). Calcium-dependent regulation of \nICS1 and EDS1 occurs through the calcium -calmodulin regulated transcription factors \nCAMTA3 and CBP60g, respectively (Wang et al., 2009b; Zhang et al., 2010). \nAmongst emerging climate risks, an increase in ambient environmental temperatures has \nbeen identified as one of the main abiotic stresses plants need to adapt to (Bita & Gerats, \n2013; Suzuki et al., 2014; Velásquez et al., 2018; Desaint et al., 2021). More specifically, \nincreases in average ambient temperatures due to climate change has significantly \naffected plant-pathogen interactions globally (Chaloner et al., 2021). It is believed that \nthese temperatures can  heighten pathogenicity by increasing phytopathogen virulence, \nfitness and/or reproduction rate (Deutsch et al., 2008; Vaumourin & Laine, 2018). In plant \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 4 \ndefence responses, this trend has generally favoured the pathogens (Garrett et al., 2006; \nDesaint et al., 2021), leading to a greater incidence and intensity of disease in crops \nincluding coffee leaf rust ( Hemileia vastatrix ), potato blight ( Phytophthora infestans ), \ncitrus canker ( Xanthomonas spp.), and wheat rust ( Puccinia spp.) (Singh et al., 2023; \nAngelotti et al., 2024). At the molecular level, an increased temperature seems to have a \ndisputed impact on PTI. One study suggest s that a short exposure to moderately high \ntemperatures ( <32°C) enhances PTI-specific transcriptomic changes  in Arabidopsis  \n(Cheng et al., 2013). Several other studies, however, have shown that PTI-dependent \nsignalling, gene expression and hormone biosynthesis are compromised by an increase \nin ambient temperature (Wang et al., 2009a; Rasmussen et al., 2013; Huot et al., 2017; \nJanda et al. , 2019; Kim et al. , 2022) . Though the impact of  an increased ambient \ntemperature on PTI has been studied extensively, this has yet to reveal its impact upon \nPTI-specific calcium signalling.  \nAs described above, calcium signalling is known to play a central role in plant defence \nsignalling. How an increase in environmental growth temperature may impact this, \nhowever, remains unknown. In this work, we use [Ca2+]cyt measurements, as a key marker \nof PTI-specific signalling, to explore the impact of an increase in ambient temperature on \nflg22-dependent responses. We initially detected a reduction in the upstream calcium \nsignalling response to flg22 following a pre -treatment at an increased temperature . \nSubsequently, we tested downstream Ca 2+ signalling by measuring the expression of \nICS1 and EDS1 and found it to be similarly compromised. Under this higher ambient \ntemperature treatment, basal FLS2 protein levels were reduced, which, at least partly, \nexplains the reduced calcium -dependent flg22 response measured following this \ntreatment. Additionally, the calcium responses (upstream and downstream) could then be \nrestored by artificially inducing FLS2 protein levels, further suggesting that reduced PTI \nin response to higher ambient temperature is due to a reduced availability of FLS2. We \nalso suggest than an increase in temperature affects FLS2 accumulation, and likely PTI, \nvia changes in membrane fluidity. This conclusion is based on the results which show that \nchemically altering membrane fluidity with benzyl alcohol (BA) similarly reduced upstream \nand downstream calcium signalling via a reduction in FLS2 protein levels. This inhibition \nof PTI by BA via reduced FLS2 levels could also be restored by the inducible expression \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 5 \nof FLS2. Our findings therefore reinforce the negative effect of an increased temperature \nupon PTI-dependent calcium signalling and dissects a potential mechanism underpinning \nthis effect.  \n \nRESULTS \nAn increase in environmental temperature reduces the upstream [Ca 2+]cyt-\ndependent response to flg22  \nTo assess the effect of an increase in environmental temperature upon plant defence \nresponses, we incubated Arabidopsis seedlings (pMAQ2) at 28°C for 24 h. This moderate \ntemperature increase (+8°C from normal growth temperature) was specifically selected \nbased on research showing that it serves as an environmental signal, rather than a stress \nstimulus (Penfield, 2008; Liu et al. , 2015) . Following this incubation, the subsequent \n[Ca2+]cyt and ROS responses to flg22 was measured. As shown in Figure 1, seedlings \npre-treated at 28°C exhibited a clear reduction in the magnitude of the calcium (1a) and \nROS (1b) response compared to those pre-treated at 20°C. This was evident in both the \nalteration of the flg22 -induced calcium signature and the significantly decreased flg22 -\nspecific area under the curve (AUC). \nMany studies have shown that an elevated environmental temperature increases \nplant membrane fluidity (Murakami et al., 2000; Martinière et al., 2011; Cano-Ramirez et \nal., 2021) . We therefore investigated whether the reduction in the flg22 -dependent \ncalcium response in seedlings incubated at 28°C was related to a temperature -induced \nincrease in membrane fluidity. To test this, seedlings were treated with benzyl alcohol \n(BA), a compound known to artificially increase membrane fluidity, for 24 h (Carratù et al., \n1996; Saidi et al., 2011; Niu & Xiang, 2018) . As shown in Figure s 1c and 1d, a BA pre-\ntreatment, like a 28°C incubation, significantly reduced the [Ca2+]cyt and ROS responses \nto flg22, respectively.   \nAn increase in environmental temperature also reduces the downstream [Ca 2+]cyt-\ndependent response to flg22  \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 6 \nHaving shown that a 24 h incubation at 28°C reduces the early signalling responses to \nflg22 (Figure 1), we next tested whether this pre-treatment also suppressed downstream \nflg22-responsive calcium-dependent gene expression. ICS1 and EDS1 are two key biotic \nstress-inducible genes involved in the production of salicylic acid (SA) during plant \ndefence responses. Both genes are also known to be regulated in a calcium -dependent \nmanner by the transcription factors CBP60g and CAMTA3, respectively  (Wang et al. , \n2009b; Zhang et al., 2010). Therefore, by using ICS1 and EDS1 expression as markers \nof downstream calcium -specific defence responses, we explored whether reducing the \nflg22-induced upstream signals with a 24 h incubation at 28°C (Figure 1), correlated with \na reduced induction of these genes. Figure 2 supports this idea, with flg22 -dependent \nICS1 and EDS1 expression clearly reduced in the seedlings pre -treated at 28°C \ncompared to those pretreated at 20°C.  \nTo test whether the impact of temperature might be due to its effect upon \nmembrane fluidity, we also measured the gene expression response to flg22 in seedlings \npre-treated at 20°C with BA. We found that the BA pre -treatment shown to reduce the \nupstream [Ca2+]cyt and ROS responses to flg22 (Figure 1), also led to a reduction in the \nflg22-dependent expression of ICS1 and EDS1 (Figure 2). The transcript level increase \nof both genes was significantly inhibited, at each time point, compared to the seedlings \npre-treated with water. Together, these data demonstrate that reduction of the upstream \ncalcium response to flg22, with either a 28°C or BA pre-treatment, correlates with reduced \nflg22-specific expression of calcium-regulated genes ICS1 and EDS1.  \nAn increase in environmental temperature , or a treatment with BA, reduce FLS2 \nlevels  \nThe results presented thus far suggest that pre-treatment at an increased environmental \ntemperature, or with BA, significantly reduces the upstream responses to flg22. To \ninvestigate the mechanism by which temperature/BA suppressed flg22 calcium -\ndependent responses, we next explored their effect on the level of the plasma membrane \nflg22 receptor, FLS2. Through western blot analysis we detected reduced  basal FLS2 \nlevels in seedlings pre -treated for 24 h at 28°C compared to those pre -treated at 20°C \n(Figure 3a). Similarly, BA pre-treatment also resulted in a clear comparative reduction in \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 7 \nFLS2 protein levels (Figure 3b). With flg22-based signalling known to be dependent upon \nFLS2, a reduced level of FLS2 in seedlings pre -treated at 28°C and with BA (Figure 3) \nmay explain why flg22 -dependent upstream [Ca 2+]cyt signalling (Figure 1) and \ndownstream gene expression (Figure 2) were also reduced in these seedlings.  \nFLS2 levels can be restored at 28°C upon inducible FLS2 over-expression  \nOur data so far suggest that a reduction in FLS2, induced by a pre -treatment at 28°C, \ncontributes to the observed suppression in the calcium -dependent flg22 response. To \nmanipulate basal FLS2 levels, we transformed pMDC7 FLS2, which allows for FLS2 \ntranscription under an estradiol-inducible (XVE) system, into wildtype pMAQ2 plants. As \na pre-treatment at 28°C (or with BA) was shown to reduce basal FLS2 levels (Figure 3), \nwe firstly investigated whether the pMDC7FLS2 construct could truly restore FLS2 protein \nlevels in these conditions. Under control conditions (no estradiol), Figure 4 supports the \nprevious findings: a reduced level of basal FLS2 is measured in wildtype seedlings pre -\ntreated at 28°C or with BA, compared to those pre-treated at 20°C. More importantly, our \ndata also show that FLS2 levels can be restored at 28°C, or with BA pre -treatment, \nfollowing an estradiol treatment, as FLS2 protein can be clearly detected under these \nconditions. This suggests that we can use the pMDC7 FLS2 construct to artificially \nincrease FLS2 levels in seedlings pre-treated at 28°C or with BA.  \n \nRestoring FLS2 levels at 28°C reinstates the calcium -dependent flg22 response at \n28°C \nAfter confirming the pMDC7 FLS2 construct restored FLS2 protein in wildtype seedlings \npre-treated at 28°C, or with BA (Figure 4), we used the pMAQ2pMDC7 FLS2 line to \ninvestigate whether this restoration recovered flg22-dependent calcium signalling. To do \nthis, we pre-treated seedlings for 24 h at 28°C  or with 30 mM BA,  together with a 16 h \ntreatment of 10 μM estradiol or 0.02% (v/v) DMSO . The [Ca2+]cyt response to flg22 was \nthen tested in both sets of seedlings. \nIn the seedlings pre-treated with DMSO, we measured a partial restoration of the flg22 -\ndependent response (Figure 5). This is seen in the characteristic flg22 [Ca2+]cyt signature \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 8 \nand an increased AUC measured in these seedlings compared to the pMAQ2 seedlings \ntreated for 24 h at 28°C, or with BA, in Figure 1. This likely reflects some basal leakiness \nof the inducible system.  Despite this, in the seedlings pre -treated with both 28 °C and \nestradiol (Figure 5a), or both BA and estradiol (Figure 5b) , an even larger increase in \nflg22-dependent calcium signalling was measured. The magnitude of the calcium \nsignature and the total AUC were both significantly higher in these seedlings, suggesting \na restoration (with estradiol) of FLS2 in pMAQ2pMDC7 FLS2 seedlings reinstates \nupstream flg22-specific calcium signalling following 28°C or BA pre-treatment \nTo determine whether restoring the upstream [Ca 2+]cyt response also restored \ndownstream signalling, we measured flg22 -responsive calcium -dependent gene \nexpression. To do this, we exposed pMAQ2pMDC7 FLS2 seedlings to pre-treatments of \n20°C, 28 °C or with BA at 20°C, together with treatments of DMSO or estradiol, and \nmeasured the ICS1 and EDS1 transcript level increases following a 3 h flg22 treatment. \nThis timing was used as it was previously shown to be within the timeframe of optimal \nflg22-dependent gene expression for both ICS1 and EDS1 (Figure 2). As shown in Figure \n6, a flg22-dependent increase in both ICS1 and EDS1 transcript level was measured in \npMAQ2pMDC7FLS2 seedlings pre -treated at 20 °C and with DMSO. This was further \nincreased in the seedlings pre -treated at 20 °C and with estradiol. This suggests the \nincreased artificial levels of FLS2 produced with the pMDC7 FLS2 construct not only \nenables the reinstatement of the calcium signature (Figure 5), but this signature is also \nfunctional in terms of regulating ICS1 and EDS1 transcription. Figure 6 also confirms \nprevious results, that a BA or 28 °C pre-treatment reduces calcium-specific flg22 -\nresponsive gene expression (see: DMSO H2O treatment). More importantly, we also show \nthat using estradiol to initiate an increase in FLS2 transcription in pMAQ2pMDC7 FLS2 \nseedlings pre -treated at 28 °C, or with BA, restores flg22 -specific ICS1 and EDS1 \nexpression (Figure 6). Taken together, we show that pre -treating wildtype seedlings at \n28°C or with BA significantly reduces the calcium -dependent response to flg22. This \nreduction, in both conditions, can be restored by using a pMDC7 FLS2 construct which \nartificially increases the levels of FLS2 in these seedlings. This in turn allows for the \nrestoration of flg22 -dependent upstream [Ca 2+]cyt signalling and downstream defense \nresponsive gene expression.  \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 9 \nDISCUSSION \nThe aim of this study was to investigate and examine the mechanistic basis of the impact \nof a moderate increase in temperature on the calcium -dependent signalling involved in \nArabidopsis pattern-triggered immunity (PTI) . To achieve this, we u sed the flg22-FLS2 \nligand-receptor pair as a PTI model, and investigated [Ca2+]cyt responses and quantified \nthe expression of calcium-regulated immunity genes, ICS1 and EDS1 following a 24h pre-\ntreatment at 28°C. We also measured basal FLS2 protein levels and tested the effect of \nmodifying these levels by using an inducible FLS2 expression system. In parallel, we also \ninvestigated the effect of  a BA treatment on the same markers of upstream and \ndownstream calcium signalling, to test whether the effects of  an increase in ambient \ntemperature might be sensed through changes in membrane fluidity.  Together, our work \nindicates that an increase in ambient temperature leads to desensitisation of PTI through \nthe reduction in the amount of active FLS2, and that the increase in temperature is likely \nsensed by plant cells through an increased membrane fluidity.  \nData in Figure 1a  and 1b  show that Arabidopsis seedlings exposed for 24  h at 28°C \ndisplay significantly reduced upstream responses to flg22, compared to those kept at \n20°C. This is seen as a clear statistically significant difference in the area under the curve \nfor the later phase of the flg22-specific calcium signature and a reduced flg22-dependent \nROS response. Very similar effects were observed when plants were pretreated for 24 h \nat 20°C  with BA (Figure 1 c and d ). How the attenuation of upstream flg22-mediated \nresponses correlate to the expression of flg22-induced calcium-regulated genes was then \ntested (Figure 2). For EDS1 (Figures b and d) and ICS1 (Figures a and c), both treatment \nat 28°C (Figures a and b) or with BA (Figures c and d) significantly inhibited  their flg22-\ninduced expression. This suggests a clear correlation between the [Ca2+]cyt response and \nthe expression of calcium-regulated genes EDS1/ICS1, and that both are reduced by a \nmoderate increase in ambient temperature or with a  treatment of BA. The similarity \nbetween the effects of 28°C and BA upon calcium signalling in response to flg22 suggests \nthat the sensing of temperature increase that leads to a reduction in sensitivity of PTI acts \nthrough plant cells assessing changes in membrane fluidity. It is known that cells remodel \nmembrane fluidity in response to changes in ambient temperature through controlling lipid \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 10 \nsaturation and fatty acid length  (Schroda et al., 2015). Literature provides a consensus \nthat this change in the physical state of the membrane can act as a “thermometer” (Niu & \nXiang, 2018; Cano -Ramirez et al. , 2021; Jung et al. , 2023) , whereby  the biophysical \nincrease in membrane fluidity caused by increases in temperature is the parameter \nsensed by cells to alert them of temperature change. Our experiments using BA, which \nwill fluidise the membrane whilst keeping a constant temperature (Pedersen & Cox, 1984; \nÖrvar et al., 2000; Sangwan et al., 2001), phenocopied the effects of 28°C treatment on \nboth cytosolic calcium responses and downstream plant immunity gene expression. This \nis consistent with the hypothesis that perception of increased ambient temperature \nleading to reduction of FLS2 levels occurs via sensing the changes in membrane fluidity.  \nPrecisely how this disruption in membrane fluidity is sensed and then relayed to effect \nflg22-dependent Ca 2+ signal is an interesting conundrum. The increase in membrane \nfluidity may act as a signal itself, detected within the plasma membrane. Although several \nion channels, notably the transient receptor potential cation channels (TRPs), have been \nshown to function as temperature sensors in animal cells (Caterina et al., 1997; Xu et al., \n2002; Vilar et al., 2020), the identity of plant temperature sensors remains largely elusive. \nSpecific calcium-permeable cyclic nucleotide gated channels (CNGCs) have been shown \nto be activated by heat and mild temperature increme nts in plants (Saidi et al., 2009; \nFinka et al., 2012; Gao et al., 2012). It may be interesting to determine the role of such \nchannels in our work, especially as specific plasma membrane bound Ca2+ channels were \nshown to be activated, and modulated, by an increase in temperature or  by chemically \nperturbating membrane fluidity  with BA (Saidi et al., 2010). It is important to remember \nthat the alteration in membrane fluidity serves also a physical cue, that may impact the \nconformation and function of plasma membrane-bound proteins and channels. Rather \nthan a signal per se , it could be that  the disruption in membrane fluidity inhibits the \nenvironment of important receptors, including FLS2.  For example, w ith several studies \nconcluding that FLS2 exists within PM nanoclusters , it could be important to measure the \neffect of changes in membrane fluidity  upon nanocluster integrity  (Bücherl et al., 2017; \nCui et al., 2018; Tran et al., 2020; Gronnier et al., 2022; Hurst et al., 2023). This aligns \nwith research showing that altering membrane composition and plasma membrane sterol \nabundance affect s FLS2-based signalling  (Cui et al. , 2018; Hurst et al. , 2023) . \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 11 \nDeciphering how the temperature -dependent impact on membrane fluidity is relayed to \nreduce Ca 2+-specific PTI signalling would reveal further insight into the mechanism \nbehind our work.  \nThough the source/nature of the signal from the membrane is currently unknown, it is still \nvery clear that an increased ambient temperature or BA treatment reduce both upstream \nand downstream calcium signalling to flg22. As FLS2 is the flg22 receptor, we investigated \nthe impact an increase in membrane fluidity has on its basal level. The specific interaction \nbetween flg22 and FLS2 has been shown to be responsible for initiating the calcium influx \nfrom external stores which creates the calcium signature we observed in Figure 1  \n(Jeworutzki et al., 2010; Ranf et al., 2011). To investigate whether an increase in ambient \ntemperature and/or BA might affect the levels of this receptor, we performed western blot \nanalysis on seedlings pre-treated for 24 h at either 28°C (Figure 3a) or with BA (Figure \n3b). This analysis clearly showed that total levels of FLS2 protein were reduced (Figure \n3) in these conditions. This suggested that the reduction in level of upstream (Figure 1) \nand downstream (Figure 2) signalling in response to fl g22 that we observed was due to \na reduction in the l evel of the primary receptor. To test this hypothesis, we generated a \nDNA construct which, when transformed into Arabidopsis, could induce FLS2 expression \nupon exogenous application of estradiol. Expression of this construct was used to see \nwhether by judiciously inducing expression of FLS2 under conditions (28°C or BA) which \nled to a reduction in total FLS2 protein seen in Figure 3 could restore calcium signalling \nin response to flg22. We first tested whether this construct could achieve increased FLS2 \nprotein levels under these conditions. As can be seen in Figure 4, under conditions of \n28°C or BA, FLS2 protein levels were reduced as already observed (Figure 3), but \nestradiol treatment led to restoration of FLS protein levels. As can be seen in Figure 5, \nwhen FLS2 was specifically expressed after 24 h of 28°C or BA at 20°C, this restored the \ncalcium response to flg22, with a significantly increased area under the curve being \nproduced. To test whether this restoration of flg22-mediated upstream calcium response \ncould also restore downstream ICS1 and EDS1 expression, we tested the same \nconditions and measured transcript levels of these 2 genes. As can be seen in Figure 6 \nwhilst 28°C and BA treatments again inhibited the flg22-mediated induction of both genes, \ncompared to expression at 20°C, restoration of FLS2 by estradiol treatment restored the \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 12 \nability of these plants to respond to flg22. These data together strongly support the idea \nthat the desensitisation of the response at both 28°C and in response to BA treatment is \ndue to a reduction in active FLS2 receptor levels. It has been shown previously that an \nacute (45 minutes) treatment at high temperature (42°C) greatly reduces the ROS \nresponse to flg22 due to reduced FLS2 (Janda et al., 2019). Our work shows that similar \neffects can be seen in response to much more moderate temperature elevations, which \nare much closer to relevant field temperatures, suggesting this phenomenon is of \nsignificant contemporary relevance to agriculture.  The impact of a smaller temperature \nincrease upon FLS2  we measured in this work  may help to explain some of the well-\nestablished research showing suppression of PTI-based signalling under more moderate \ntemperature increases. For example, FLS2-dependent callose depos ition (Gómez-\nGómez & Boller, 2000; Zipfel et al. , 2004)  in response to flg22 is reduced in plants \nexposed to a 24 h pre-treatment at 37°C (Janda et al., 2019) or 48 h pre -treatment at \n30°C (Huot et al. , 2017) . Even more related to our work, t ranscriptomic analysis has \npreviously shown a reduction in flg22-dependent ICS1 and EDS1 gene expression \nfollowing a short pre -treatment at 37°C  (Rasmussen et al., 2013). This suppression of \npathogen-induced ICS1 expression has also been measured following both a 30°C (Huot \net al., 2017),  and 28°C (Shields et al. , 2025)  treatment, which correlate s with the \nsubsequent reduction of salicylic acid production at 28°C compared to 23°C  (Kim et al., \n2022). This work clearly aligns with the data in our study, suggesting th e suppression of \nFLS2 at 28°C may be involved in a more global suppression of flg22 -dependent \nresponses following increased ambient temperatures. Though it is clear the temperature-\nspecific reduction of FLS2 strongly impairs PTI signalling, in the future we will investigate \nthe mechanism by which FLS2 levels are reduced in response to temperature and BA. It \nis most likely that these treatments impose destabilisation of the protein and protein \ndegradation, therefore measuring ubiquitination of FLS2 in response to 28°C/ BA would \nbe a good approach. Equally, it is possible that mechanisms that have been described for \nautophagic regulation of FLS2 could be involved (Yang et al., 2019). Increases in ambient \ntemperature are well-known inducers of autophagy (Sedaghatmehr et al., 2019) and so it \nwould be interesting if the ORM1/ORM2 orosomucoid proteins which selectively degrade \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 13 \nFLS2 through ATG8 autophagy (Yang et al., 2019), are increased in expression or activity \nin response to increased ambient temperature. \nOverall, the work described here demonstrates that a potential component in the increase \nin balance of power in favour of pathogens of crops due to modest increases in \ntemperature during climate change could be due to destabilisation of receptors evolved \nto detect pathogens. Understanding this, and future research into the mechanisms by \nwhich these receptors are regulated by increases in temperature might be useful for \nreading/engineering crops with robustness of receptor levels under increasing \ntemperature. Targets could be the components of the ubiquitin pathway specifically \nregulating this phenomenon, the temperature -dependent autophagy pathway leading to \nFLS2 or membrane micro domain and lipid species that govern FLS2 stability at elevated \ntemperature. In addition, it will be interesting to expand this work to investigate whether \nthe reduction in FLS2 levels is seen similarly in other PRRs.  \n \nMATERIAL AND METHODS \nPlant materials and growth conditions \nAll seedlings used were in the Arabidopsis thaliana (A. thaliana ) Col -0 ecotype \nbackground. Wildtype calcium measurement experiments were performed on transgenic \nseedlings constitutively expressing the calcium reporter 35S::apoaequorin in the cytosol \n(Col-0pMAQ2) (Knight et al. , 1991) . The fls2-26pMAQ2 mutant, which constitutively \nexpresses cytosolic 35S::apoaequorin, and possesses a nucleotide missense mutation \n(Q865*) in FLS2, was described previously (Ranf et al. , 2012) . The fls2C mutant \n(SAIL_691C4) was used as a FLS2 null mutant in the immunoblotting work (Zipfel et al., \n2004). The pMAQ2pMDC7FLS2 line used was created in this work. Seeds were surface \nsterilised in 70% ethanol (v/v) and sown onto 1 × Murashige and Skoog (MS; Duchefa \nBiochemie BV, Haarlem, Netherlands) medium, pH 5.8, 0.8% (w/v) plant tissue culture \nagar (Sigma-Aldrich, St Louis, MO, USA) and stratified at 4°C in darkness for 48 h. Plants \nwere then grown in a Percival CU -36L5D growth chamber (CLF PlantClimatics, \nEmersacker, Germany) at 20±1°C, with a light intensity of 150 μmol m-2 s-1 and a 16 h \nlight/8 h dark ph otoperiod. Pre -treatments were conducted 24 h before the start of \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 14 \nexperiments with specific details given below. All experiments were performed on 14-day-\nold seedlings.  \nProducing the pMDC7FLS2pMAQ2 line \nThe pENTRTM/D-TOPOTM entry vector (Thermo Fisher Scientific, Loughborough, UK) was \nlinearised using NotI and AscI restriction enzymes (New England Biolabs, UK), and the \nresulting product isolated from an agarose gel by gel extraction (QIAGEN Ltd., UK). Three \nsynthetic gene bl ocks, designed to span across the full FLS2 coding sequence (CDS), \nwere ordered from IDT (Integrated DNA Technologies, Leuven, Belgium). The sequences \nof these gene blocks can be found in Supplementary Table 1. A Gibson Assembly® \nCloning Kit (NEB, Cat. No E5510S) was used to assemble the FLS2CDS gene fragments \ninto the linearised vector according to the manufacturer’s instructions. The binary \ndestination vector pMDC7, containing the estradiol inducible XVE system, was described \npreviously (Curtis & Grossniklaus, 2003) . Gateway recombination using the LR \nClonaseTM II Enzyme Mix (Life Technologies, Paisley, UK) was then performed between \nthe pENTRFLS2CDS entry clone and the pMDC7 destination vector.  \nIn vivo reconstitution of aequorin and pre-treatment conditions \nFor calcium measurements, 24 h before the start of measurements 13-day-old seedlings \nwere floated on a 5 mL H 2O solution in 6 -well plates. For temperature pre -treatment \nconditions, the seedlings were moved into one of two identical Sanyo MLR -351 growth \ncabinets (Sanyo Electric co. Ltd, Moriguchi, Japan). One growth cabinet was set at 20°C, \nand the other was set at 28 °C. For benzyl alcohol (BA) pre -treatment conditions, \nseedlings were floated in either 5 mL H2O or 30 mM BA and placed inside the 20°C growth \ncabinet. Aequorin reconstitution was performed during these pre-treatment conditions by \nadding coelenterazine (final concentration 10 μM in 1% (v/v MeOH) to the solution 16 h \nbefore the start of experiments. Where used, estradiol pre-treatment was also conducted \nduring this period by adding estradiol (final concentration: 10 μM in 0.02% (v/v) DMSO) \nor DMSO (final concentration 0.02%(v/v)) as a control to the solution 16 h before the start \nof the experiment. \n \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 15 \n \n[Ca2+]cyt-dependent luminescence measurements  \nFollowing pre-treatment, individual seedlings were transferred into 3.5 mL luminometer \ncuvettes (Sarstedt, Nümbrecht, Germany) containing 0.5 mL H 2O. After a 30 min period \nof rest, individual cuvettes were then inserted into the luminometer sample housing. \nLuminescence levels were recorded every 1 s using a digital chemiluminometer with \ndiscriminator and cooled housing unit (Electron Tubes Limited, Middlesex, UK) to reduce \nbackground noise (Knight et al., 1991). Luminescence was recorded for 60 s before the \ninjection of 0.5 mL 1 μM flg22 (QRLSTGSRINSAKDDAAGLQIA) (GenScript Biotech, New \nJersey, USA). The subsequent changes in luminescence were recorded for a further 240 \ns. A 300 s discharge was performed at the end of the experiment by injecting equal (1 \nmL) volume of 2M CaCl 2, 20% (v/v) ethanol. Calibration was performed as described \npreviously (Knight et al., 1991). \nROS burst measurements  \nROS burst measurements were performed on whole seedlings using an adapted version \nof a previously described protocol (Kadota et al., 2014). Briefly, individual seedlings were \nincubated overnight in luminometer cuvettes containing 17 μg/mL luminol (Sigma-Aldrich) \nand 20 μg/mL horseradish peroxidase (HRP, Sigma -Aldrich). The following day, the \nsolution was replaced with a 0.5 μM flg22 solution (in 17 μg/mL luminol, 20 μg/mL HRP) \nand the individual cuvettes were then inserted into the luminometer sample housing. \nLuminescence levels were recorded every 1 s using a digital chemiluminometer with \ndiscriminator and cooled housing unit (Electron Tubes Limited, Middlesex, UK) to reduce \nbackground noise (Knight et al., 1991). Luminescence levels were recorded for 1500 s \nand ROS production is displayed as total luminescence recorded.  \nRNA extraction, cDNA preparation and gene expression measurements \nPre-treatments were performed as described above for gene expression experiments. \nFor treatments, 1 mL of solution was removed from each condition and 1 mL flg22 (or \nH2O) at 5 × concentration was added (in either 0 or 150 mM BA) and the seedlings were \nreturned to either the 20°C or 28°C growth cabinet until harvested. Tissue was harvested \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 16 \n1, 3 and 6 h after the treatment. For each sample, representing one condition at a single \ntime point, 15 seedlings were pooled together for subsequent RNA extraction. A high -\ncapacity cDNA reverse transcriptase kit (Applied Biosystems, Foster City, CA, USA) was \nused to reverse transcribe total RNA (2 μg) obtained with an RNeasy ReliaPrep ™ RNA \nMiniprep System Plant Total RNA kit (Promega, Southampton, UK). Quantitative real-time \nPCR was performed using 5 μL of 1:50 diluted cDNA in a total volume of 15 μL using an \nApplied Biosystem 7300 real time PCR machine. Relative expression of  Enhanced \nDisease Susceptibility 1  (EDS1) ( At3g48090) and  Isochorismate Synthase 1  (ICS1) \n(At1g74710) were measured with Fast Start SYBR Green Master Mix with ROX  \n(Promega, Southampton, UK) using the following primers:  EDS1 Fw 5′ -\nACCTAACCGAGCGCTATCAC-3′, EDS1 Rev 5′-TTGTCCGGATCGAAGAAATC-3′, ICS1 \nFw 5′ -CAAATCTCAACCTCCGTCGT-3′, ICS1 Rev 5′ -AATCAATTGCTCCGATTTGC-3′. \nLevels were normalised to the levels of the endogenous  PEX4 housekeeping gene \n(At5g25760), using the following primers:  PEX4 Fw 5′ -\nTCATAGCATTGATGGCTCATCCT-3′ and PEX4 Rev 5′ -\nACCCTCTCACATCACCAGATCTTAG-3′. Relative quantification was performed using \nthe delta cycle threshold ( ΔΔCt) method (Livak & Schmittgen, 2001)  and the values \nobtained representing the relative quantification (RQ) were calculated as described \npreviously (Knight et al., 2009).  \n \nGeneration of anti-FLS2 antibodies \nAnti-FLS2 antibodies were generated against a peptide targeting the C-terminus of FLS2 \n(CKANSFREDRNEDREV) and coupled to keyhole limpet hemocyanin for immunisation. \nPeptide synthesis, conjugation, and immunisations were performed by GenScript. Rabbit \nimmune serum was used for affinity purification against bead -coupled antigen peptide. \nThe specificity of affinity purified antibody was confirmed by immunoblotting against \nprotein extracts from Arabidopsis seedlings lacking FLS2 protein (Supplementary Figure \n1). \n \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 17 \nSDS-PAGE and western blotting \nPre-treatments were performed as described above and samples were harvested directly \nafter the 24 h pre -treatment (no flg22 treatment). For each sample, representing one \ncondition, 15 seedlings were pooled together for subsequent protein extraction. Seedlings \nwere flash frozen in liquid nitrogen and thoroughly homogenised in equal volume of \nextraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 2 mM EDTA), \n0.002M DTT, 1% (v/v) Igepal, and 1 × Phosphatase (P5726) and 1 × Protease (P9599) \nInhibitors, Sigma-Aldrich). The samples were left on ice for 30 min to solubilise membrane \nproteins before being centrifuged at 20,000 g for 20 min at 4°C. Each resulting 100 μ L \nsample was normalised to contain the same amount of protein, and 35 μL 5 × SDS-PAGE \nloading buffer (10% SDS (w/v), 50% glycerol, 300 mM Tris -HCl pH 6.8, 0.125% (w/v) \nbromophenol blue) and 15 μL 1M DTT was added to each. Protein samples were heated \nto 90°C for 10 min prior to electrophoresis.  \n Samples were loaded onto 8% SDS -PAGE gels and electrophoresis was \nperformed (in 25 mM Tris, 192 mM glycine, 0.1% SDS) at 150 V for approximately 2 h. \nSubsequently, proteins were transferred at 4°C onto activated PVDF membranes using \nwet transfer (in 25 mM  Tris, 192 mM glycine, 20% (v/v) MeOH) at 30 V for 90 min. \nMembranes were subsequently blocked overnight with 5% (w/v) skimmed milk powder \ndissolved in fresh Tris buffered saline containing Tween -20 (TBS-T; 10 mM Tris-HCl pH \n8, 150 mM NaCl, 0.1% (v/v) Tween-20) at 4°C with gentle (40 RPM) agitation. The primary \nantibody (α-FLS2, KLH-conjugated) was then added at a 1:2,500 dilution in TBS -T (5% \n(w/v) skimmed milk) to the membrane. The membrane was then incubated for 2 h at room \ntemperature with gentle (40 RPM) agitation. After primary antibody incubation the \nmembrane was wash ed five times with fresh TBS -T for 10 min each. The secondary \nantibody (goat anti-mouse IgG (H/L) HRP polyclonal antibody, Bio-Rad, USA) was added \nat a 1:5,000 dilution in TBS -T (5% (w/v)  skimmed milk) to the membrane. Incubation \noccurred at room temperature with gentle (40 RPM) agitation for 1 h and the membrane \nwas washed again with fresh TBS-T before detection. \n Western blots were visualised using the SuperSignalTM West Femto Detection Kit \n(Thermo Fisher Scientific, Loughborough, UK) according to the manufacturer’s \ninstructions. The substrate was distributed equally over the membrane and the HRP -\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 18 \ndependent chemiluminescence was detected using either a ChemiDoc Imaging System \n(Bio-Rad, California, USA) or a photon counting camera.  \n \nAUTHOR CONTRIBUTIONS \nBCICJ and MRK designed the research, BCICJ, KWB and ES performed the \nexperimental work, BCICJ performed the data analyses, collection and interpretation and \nBCICJ, MRK, KWB and CZ contributed to writing.  \n \nACKNOWLEDGEMENTS \nThis research was funded by  the Biotechnology and Biological Sciences Research \nCouncil (BBSRC) through the award of a DTP PhD studentship (ref 2182091) to BCICJ , \nand core funding to CZ provided by the University of Zurich and the Gatsby Charitable \nFoundation. We would like to thank Stefanie Ranf for providing fls2-26pMAQ2 and Ueli \nGrossniklaus for the pMDC7 binary vecto r. We also  acknowledge and  thank the help \ngiven by Julia Davies. \n \nREFERENCES \nAngelotti F, Hamada E, Bettiol W (2024). A Comprehensive Review of Climate Change \nand Plant Diseases in Brazil. Plants 13: 2447. \nBita CE, Gerats T (2013). Plant tolerance to high temperature in a changing environment: \nScientific fundamentals and production of heat stress -tolerant crops. Frontiers in Plant \nScience 4: 273. \nBücherl CA, Jarsch IK, Schudoma C, Segonzac C, Mbengue M, Robatzek S, \nMacLean D, Ott T, Zipfel C (2017). Plant immune and growth receptors share common \nsignalling components but localise to distinct plasma membrane nanodomains. eLife 6: \ne25114. \nCano-Ramirez DL, Carmona -Salazar L, Morales -Cedillo F, Ramírez -Salcedo J, \nCahoon EB, Gavilanes -Ruíz M  (2021). Plasma Membrane Fluidity: An Environment \nThermal Detector in Plants. Cells 10: 2778. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 19 \nCarratù L, Franceschelli S, Pardini CL, Kobayashi GS, Horvath I, Vigh L, Maresca B \n(1996). Membrane lipid perturbation modifies the set point of the temperature of heat \nshock response in yeast. Proceedings of the National Academy of Sciences of the United \nStates of America 93: 3870. \nCaterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D (1997). \nThe capsaicin receptor: A heat -activated ion channel in the pain pathway. Nature 389: \n816–824. \nChaloner TM, Gurr SJ, Bebber DP  (2021). Plant pathogen infection risk tracks global \ncrop yields under climate change. Nature Climate Change 2021 11: 710–715. \nCheng C, Gao X, Feng B, Sheen J, Shan L, He P  (2013). Plant immune response to \npathogens differs with changing temperatures. Nature Communications 4: 2530. \nCui Y, Li X, Yu M, Li R, Fan L, Zhu Y, Lin J (2018). Sterols regulate endocytic pathways \nduring flg22-induced defense responses in Arabidopsis. Development 145. \nCurtis MD, Grossniklaus U (2003). A Gateway Cloning Vector Set for High-Throughput \nFunctional Analysis of Genes in Planta. Plant Physiology 133: 462–469. \nDesaint H, Aoun N, Deslandes L, Vailleau F, Roux F, Berthomé R (2021). Fight hard \nor die trying: when plants face pathogens under heat stress. New Phytologist 229: 712–\n734. \nDeutsch CA, Tewksbury JJ, Huey RB, Sheldon KS, Ghalambor CK, Haak DC, Martin \nPR (2008). Impacts of climate warming on terrestrial ectotherms across latitude. \nProceedings of the National Academy of Sciences of the United States of America  105: \n6668–6672. \nFelix G, Duran JD, Volko S, Boller T (1999). Plants have a sensitive perception system \nfor the most conserved domain of bacterial flagellin. Plant Journal 18: 265–276. \nFinka A, Cuendet AFH, Maathuis FJM, Saidi Y, Goloubinoff P  (2012). Plasma \nmembrane cyclic nucleotide gated calcium channels control land plant thermal sensing \nand acquired thermotolerance. Plant Cell 24: 3333–3348. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 20 \nGao F, Han X, Wu J, Zheng S, Shang Z, Sun D, Zhou R, Li B (2012). A heat-activated \ncalcium-permeable channel - Arabidopsis cyclic nucleotide -gated ion channel 6 - Is \ninvolved in heat shock responses. Plant Journal 70: 1056–1069. \nGarrett KA, Dendy SP, Frank EE, Rouse MN, Travers SE  (2006). Climate change \neffects on plant disease: Genomes to ecosystems. Annual Review of Phytopathology 44: \n489–509. \nGómez-Gómez L, Boller T (2000). FLS2: An LRR Receptor–like Kinase Involved in the \nPerception of the Bacterial Elicitor Flagellin in Arabidopsis. Molecular Cell 5: 1003–1011. \nGronnier J, Franck CM, Stegmann M, Defalco TA, Abarca A, von Arx M, Dünser K, \nLin W, Yang Z, Kleine -Vehn J, et al.  (2022). Regulation of immune receptor kinase \nplasma membrane nanoscale organization by a plant peptide hormone and its receptors. \neLife 11: e74162. \nHashimoto K, Kudla J  (2011). Calcium decoding mechanisms in plants. Biochimie 93: \n2054–2059. \nHuot B, Castroverde CDM, Velásquez AC, Hubbard E, Pulman JA, Yao J, Childs KL, \nTsuda K, Montgomery BL, He SY (2017). Dual impact of elevated temperature on plant \ndefence and bacterial virulence in Arabidopsis. Nature Communications 8: 1808. \nHurst CH, Turnbull D, Xhelilaj K, Myles S, Pflughaupt RL, Kopischke M, Davies P, \nJones S, Robatzek S, Zipfel C, et al.  (2023). S-acylation stabilizes ligand -induced \nreceptor kinase complex formation during plant pattern -triggered immune signaling. \nCurrent Biology 33: 1588-1596.e6. \nJanda M, Lamparová L, Zubíková A, Burketová L, Martinec J, Krčková Z  (2019). \nTemporary heat stress suppresses PAMP-triggered immunity and resistance to bacteria \nin Arabidopsis thaliana. Molecular Plant Pathology 20: 1005–1012. \nJeworutzki E, Roelfsema MRG, Anschütz U, Krol E, Elzenga JTM, Felix G, Boller T, \nHedrich R, Becker D (2010). Early signaling through the arabidopsis pattern recognition \nreceptors FLS2 and EFR involves Ca 2+-associated opening of plasma membrane anion \nchannels. Plant Journal 62: 367–378. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 21 \nJones JDG, Dangl JL (2006). The plant immune system. Nature 444: 323–329. \nJung JH, Seo PJ, Oh E, Kim J (2023). Temperature perception by plants. Trends in Plant \nScience 28: 924–940. \nKadota Y, Sklenar J, Derbyshire P, Stransfeld L, Asai S, Ntoukakis V, Jones JD, \nShirasu K, Menke F, Jones A, et al. (2014). Direct Regulation of the NADPH Oxidase \nRBOHD by the PRR -Associated Kinase BIK1 during Plant Immunity. Molecular Cell 54: \n43–55. \nKim JH, Castroverde CDM, Huang S, Li C, Hilleary R, Seroka A, Sohrabi R, Medina-\nYerena D, Huot B, Wang J, et al. (2022). Increasing the resilience of plant immunity to \na warming climate. Nature 607: 339–344. \nKnight MR, Campbell AK, Smith SM, Trewavas AJ (1991). Transgenic plant aequorin \nreports the effects of touch and cold -shock and elicitors on cytoplasmic calcium. Nature \n352: 524–526. \nKnight H, Mugford SG, Ülker B, Gao D, Thorlby G, Knight MR (2009). Identification of \nSFR6, a key component in cold acclimation acting post -translationally on CBF function. \nPlant Journal 58: 97–108. \nKudla J, Becker D, Grill E, Hedrich R, Hippler M, Kummer U, Parniske M, Romeis T, \nSchumacher K  (2018). Advances and current challenges in calcium signaling. New \nPhytologist 218: 414–431. \nLenzoni G, Liu J, Knight MR  (2018). Predicting plant immunity gene expression by \nidentifying the decoding mechanism of calcium signatures. New Phytologist 217: 1598–\n1609. \nLiu J, Feng L, Li J, He Z (2015). Genetic and epigenetic control of plant heat responses. \nFrontiers in Plant Science 6: 133426. \nLivak KJ, Schmittgen TD (2001). Analysis of relative gene expression data using real -\ntime quantitative PCR and the 2-ΔΔCt method. Methods 25: 402–408. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 22 \nMartinière A, Shvedunova M, Thomson AJW, Evans NH, Penfield S, Runions J, \nMcwatters HG  (2011). Homeostasis of plasma membrane viscosity in fluctuating \ntemperatures. New Phytologist 192: 328–337. \nMcAinsh MR, Hetherington AM (1998). Encoding specificity in Ca2+ signalling systems. \nTrends in Plant Science 3: 32–36. \nMcAinsh MR, Pittman JK (2009). Shaping the calcium signature. New Phytologist 181: \n275–294. \nMurakami Y, Tsuyama M, Kobayashi Y, Kodama H, Iba K (2000). Trienoic fatty acids \nand plant tolerance of high temperature. Science 287: 476–479. \nNiu Y, Xiang Y (2018). An Overview of Biomembrane Functions in Plant Responses to \nHigh-Temperature Stress. Frontiers in Plant Science 9: 915. \nÖrvar BL, Sangwan V, Omann F, Dhindsa RS  (2000). Early steps in cold sensing by \nplant cells: The role of actin cytoskeleton and membrane fluidity. Plant Journal 23: 785–\n794. \nPedersen JZ, Cox RP  (1984). Relationship between Thylakoid Membrane Fluidity and \nthe Kinetics of Salt Induced Fluorescence Changes: a Spin Label Study. Advances in \nPhotosynthesis Research: 51–54. \nPenfield S  (2008). Temperature perception and signal transduction in plants. New \nPhytologist 179: 615–628. \nPoovaiah BW, Du L  (2018). Calcium signaling: decoding mechanism of calcium \nsignatures. New Phytologist 217: 1394–1396. \nRanf S, Eschen -Lippold L, Pecher P, Lee J, Scheel D  (2011). Interplay between \ncalcium signalling and early signalling elements during defence responses to microbe- or \ndamage-associated molecular patterns. Plant Journal 68: 100–113. \nRanf S, Grimmer J, Pöschl Y, Pecher P, Chinchilla D, Scheel D, Lee J  (2012). \nDefense-related calcium signaling mutants uncovered via a quantitative high -throughput \nscreen in Arabidopsis thaliana. Molecular Plant 5: 115–130. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 23 \nRasmussen S, Barah P, Suarez-Rodriguez MC, Bressendorff S, Friis P, Costantino \nP, Bones AM, Nielsen HB, Mundy J (2013). Transcriptome Responses to Combinations \nof Stresses in Arabidopsis. Plant Physiology 161: 1783–1794. \nSaidi Y, Finka A, Goloubinoff P  (2011). Heat perception and signalling in plants: A \ntortuous path to thermotolerance. New Phytologist 190: 556–565. \nSaidi Y, Finka A, Muriset M, Bromberg Z, Weiss YG, Maathuis FJM, Goloubinoff P  \n(2009). The heat shock response in moss plants is regulated by specific calcium -\npermeable channels in the plasma membrane. Plant Cell 21: 2829–2843. \nSaidi Y, Peter M, Fink A, Cicekli C, Vigh L, Goloubinoff P  (2010). Membrane lipid \ncomposition affects plant heat sensing and modulates Ca 2+-dependent heat shock \nresponse. Plant Signaling & Behavior 5: 1530–1533. \nSangwan V, Foulds I, Singh J, Dhindsa RS  (2001). Cold-activation of Brassica napus \nBN115 promoter is mediated by structural changes in membranes and cytoskeleton, and \nrequires Ca2+ influx. Plant Journal 27: 1–12. \nSchroda M, Hemme D, Mühlhaus T (2015). The Chlamydomonas heat stress response. \nPlant Journal 82: 466–480. \nSedaghatmehr M, Thirumalaikumar VP, Kamranfar I, Marmagne A, Masclaux -\nDaubresse C, Balazadeh S  (2019). A regulatory role of autophagy for resetting the \nmemory of heat stress in plants. Plant Cell and Environment 42: 1054–1064. \nShields A, Yao L, Rossi CAM, Collado Cordon P, Kim JH, AlTemen WMA, Li S, \nMarchetta EJR, Shivnauth V, Chen T, et al. (2025). Warm temperature suppresses plant \nsystemic acquired resistance by intercepting N -hydroxypipecolic acid biosynthesis. The \nPlant Journal 123: e70374. \nSingh BK, Delgado -Baquerizo M, Egidi E, Guirado E, Leach JE, Liu H, Trivedi P . \n(2023). Climate change impacts on plant pathogens, food security and paths forward. \nNature Reviews Microbiology 21: 640–656. \nSuzuki N, Rivero RM, Shulaev V, Blumwald E, Mittler R (2014). Abiotic and biotic stress \ncombinations. New Phytologist 203: 32–43. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 24 \nTran TM, Ma Z, Triebl A, Nath S, Cheng Y, Gong BQ, Han X, Wang J, Li JF, Wenk MR, \net al. (2020). The bacterial quorum sensing signal DSF hijacks Arabidopsis thaliana sterol \nbiosynthesis to suppress plant innate immunity. Life science alliance 3. \nVaumourin E, Laine AL  (2018). Role of temperature and coinfection in mediating \npathogen life-history traits. Frontiers in Plant Science 871: 408482. \nVelásquez AC, Castroverde CDM, He SY  (2018). Plant–Pathogen Warfare under \nChanging Climate Conditions. Current Biology 28: R619–R634. \nVilar B, Tan CH, McNaughton PA  (2020). Heat detection by the TRPM2 ion channel. \nNature 584: E5–E12. \nWang Y, Bao Z, Zhu Y, Hua J  (2009a). Analysis of temperature modulation of plant \ndefense against biotrophic microbes. Molecular Plant-Microbe Interactions 22: 498–506. \nWang L, Tsuda K, Sato M, Cohen JD, Katagiri F, Glazebrook J  (2009b). Arabidopsis \nCaM Binding Protein CBP60g Contributes to MAMP -Induced SA Accumulation and Is \nInvolved in Disease Resistance against Pseudomonas syringae . PLOS Pathogens  5: \ne1000301. \nXu H, Ramsey IS, Kotecha SA, Moran MM, Chong JA, Lawson D, Ge P, Lilly J, Silos-\nSantiago I, Xie Y, et al. (2002). TRPV3 is a calcium -permeable temperature-sensitive \ncation channel. Nature 418: 181–186. \nYang F, Kimberlin AN, Elowsky CG, Liu Y, Gonzalez-Solis A, Cahoon EB, Alfano JR \n(2019). A Plant Immune Receptor Degraded by Selective Autophagy. Molecular Plant 12: \n113–123. \nZhang Y, Xu S, Ding P, Wang D, Cheng YT, He J, Gao M, Xu F, Li Y, Zhu Z, et al. \n(2010). Control of salicylic acid synthesis and systemic acquired resistance by two \nmembers of a plant -specific family of transcription factors. Proceedings of the National \nAcademy of Sciences of the United States of America 107: 18220–18225. \nZipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JDG, Felix G, Boller T  (2004). \nBacterial disease resistance in Arabidopsis through flagellin perception. Nature 428: 764–\n767. \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 25 \n \nFIGURE LEGENDS \nFigure 1. Flg22-induced [Ca2+]cyt and ROS increases are quantitatively reduced in \nArabidopsis seedlings pre-treated at 28ºC or with benzyl alcohol (BA). [Ca2+]cyt and \nROS changes in response to 0.5 μM flg22 in Arabidopsis seedlings pre -treated for 24 h \na) and b) at 20ºC or 28ºC or c) and d) at 20ºC with 0mM (H 2O) or 30mM BA. The traces \nshown are means of 15 replicate measurements and the error bars represent the S.E.M. \nFor a) and c) the average area under the curve (AUC  (µM)) ± S.E.M (SE) values for the \nflg22-induced calcium responses (100-300 s) are shown. The AUC values are the means \nof 15 replicate responses and the p value shown is the significance of the differences in \nthe AUC as determined by a pairwise t-test. For b) and d) the average total ROS \nproduction (∑ROS (RLU)) ± S.E.M (SE) values for the flg22-induced ROS responses (0-\n1500 s) are shown. The ∑ROS values are the means of 15 replicate responses and the \np value shown is the significance of the differences in the ∑ROS as determined by a \npairwise t-test. \n \nFigure 2. Flg22 -dependent ICS1 and EDS1 expression is reduced in Arabidopsis \nseedlings pre-treated at 28°C or with benzyl alcohol (BA). Measurement by qPCR of \nthe fold increases in a) and c) ICS1 and b) and d) EDS1 transcript expression in \nArabidopsis seedlings pre-treated for 24 h at a) and b) 20°C or 28°C, or c) and d) at 20°C \nwith 0 mM (H2O) or 30 mM benzyl alcohol (BA), in response to water or 0.5 μM flg22 1, \n3, and 6 h after the start of treatment. Relative Quantification (RQ) values were calculated \nafter normalisation to PEX4 expression levels. The value produced for each treatment is \nthe mean of three biological replicates, with each biological replicate representing the \nmean value of three technical repeats. Error bars represent the S.E.M and significant \ndifferences (p ≤0.05) were determined using ANOVA and the Tukey HSD post hoc test at \na 95% confidence interval. Bars with the same letter are not significantly different. \n \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 26 \nFigure 3. Basal FLS2 protein levels are lower in Arabidopsis seedlings pre -treated \nat 28°C or with benzyl alcohol (BA). Total protein was extracted from 14-day old pMAQ2 \nor fls2C (control) Arabidopsis seedlings pre-treated at a) 20°C or 28°C or b) at 20°C with \n0 mM or 30 mM benzyl alcohol (BA) for 24 h. FLS2 levels were detected by western \nblotting. After detection, the blots were stained with Coomassie brilliant blue (CBB) to \ndisplay loading. Values indicate the size (KDa) of the bands with the expected size of \nFLS2 indicated on the blot. Each sample loaded onto the blot is a biological replicate.  \n \nFigure 4. Basal FLS2 protein levels are restored in Arabidopsis seedlings pre -\ntreated at 28°C or with benzyl alcohol (BA) using the pMDC7 FLS2 construct. Total \nprotein was extracted from 14-day old pMAQ2pMDC7FLS2 or fls2C (control) Arabidopsis \nseedlings pre-treated for 24 h at 20°C, 28°C or with 30 mM benzyl alcohol (BA). Seedlings \nalso underwent a 16 h treatment of 10 μM estradiol (+) or 0.02% (v/v) DMSO ( -) before \nprotein extraction. FLS2 levels were detected by western blotting. After detection, the \nblots were stained with Coomassie brilliant blue (CBB) to display loading. Values indicate \nthe size (KDa) of the bands with the expected size of FLS2 indicated on the blot.  \n \nFigure 5. Flg22 -induced [Ca2+]cyt increases are restored in Arabidopsis seedlings \npre-treated at 28°C or with benzyl alcohol (BA) using the pMDC7 FLS2 construct. \n[Ca2+]cyt changes in response to 0.5 μM flg22 in pMAQ2pMDC7 FLS2 Arabidopsis \nseedlings pre-treated for 24 h a) 28°C or b) at 20°C with 30 mM BA and concurrently pre-\ntreated for 16 h with 10 μM estradiol (EST) or 0.02% (v/v) DMSO. The traces shown are \nmeans of 15 replicate measurements and the error bars represent the S.E.M. The \naverage area under the  curve (AUC  (µM)) ± S.E.M (SE) values for the flg22 -induced \ncalcium responses (100-300 s) are shown. The AUC values are the means of 15 replicate \nresponses and the p value shown is the significance of the differences in the AUC as \ndetermined by a pairwise t-test.  \n \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 27 \nFigure 6. Flg22 -dependent ICS1 and EDS1 expression is restored in Arabidopsis \nseedlings pre-treated at 28°C or with benzyl alcohol (BA) using the pMDC7 FLS2 \nconstruct. Measurement by qPCR of the fold increases in a) ICS1 and b) EDS1 transcript \nexpression in pMAQ2pMDC7 FLS2 Arabidopsis seedlings pre -treated for 24 h at 20 °C, \n28°C or with 30 mM benzyl alcohol (BA) in response to water or 0.5 μM flg22 3 h after the \nstart of treatment. The seedlings were also concurrently pre -treated for 16 h with 10 μM \nestradiol or 0.02% (v/v) DMSO. Relative Quantification (RQ) values were calculated after \nnormalisation to PEX4 expression levels. The value produced for each treatment is the \nmean of three biological replicates, with each biological replicate representing the mean \nvalue of three technical repeats. Error bars represent the S.E.M and significant \ndifferences (p ≤0.05) were determined using ANOVA and the Tukey HSD post hoc test at \na 95% confidence interval. Bars with the same letter are not significantly different. \n  \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 28 \n \nFigure 1. Flg22-induced [Ca2+]cyt and ROS increases are quantitatively reduced in \nArabidopsis seedlings pre-treated at 28ºC or with benzyl alcohol (BA). [Ca2+]cyt and \nROS changes in response to 0.5 μM flg22 in Arabidopsis seedlings pre -treated for 24 h \na) and b) at 20ºC or 28ºC or c) and d) at 20ºC with 0mM (H 2O) or 30mM BA. The traces \nshown are means of 15 replicate measurements and the error bars represent the S.E.M. \nFor a) and c) the average area under the curve (AUC  (µM)) ± S.E.M (SE) values for the \nflg22-induced calcium responses (100-300 s) are shown. The AUC values are the means \nof 15 replicate responses and the p value shown is the significance of the differences in \nthe AUC as determined by a pairwise t-test. For b) and d) the average total ROS \nproduction (∑ROS (RLU)) ± S.E.M (SE) values for the flg22-induced ROS responses (0-\n1500 s) are shown. The ∑ROS values are the means of 15 replicate responses and the \np value shown is the significance of the differences in the ∑ROS as determined by a \npairwise t-test. \n(a)\n0\n0.1\n0.2\n0.3\n0.4\n0.5\n0.6\n0.7\n0 50 100 150 200 250 300\n[Ca2+]cyt (µM)\nTime (s)\n20ºC 28ºC\n28ºC20ºC\n10.36\n±1.17\n21.39\n±1.96\nAUC (µM) \n±SE\n<0.01p value\n0\n0.1\n0.2\n0.3\n0.4\n0.5\n0.6\n0.7\n0 50 100 150 200 250 300\n[Ca2+]cyt (µM)\nTime (s)\n0mM BA 30mM BA\n30 mM BA0 mM BA\n9.26\n±1.6\n19.18\n±1.85\nAUC (µM) \n± SE\n<0.01p value\n(c)\n0\n400\n800\n1200\n1600\n2000\n0 300 600 900 1200 1500\nLuminescence (RLU) \nTime (s)\n0 mM BA 30 mM BA\n0\n400\n800\n1200\n1600\n2000\n0 300 600 900 1200 1500\nLuminescence (RLU)\nTime (s)\n20ºC 28ºC\n28ºC20ºC\n150391\n±21689\n207950\n±20593\n∑ROS (RLU) \n±SE\n<0.05p value\n30 mM BA0 mM BA\n117365\n±14493\n241522\n±23267\n∑ROS \n(RLU) ± SE\n<0.001p value\n(b)\n(d)\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 29 \n \nFigure 2. Flg22 -dependent ICS1 and EDS1 expression is reduced in Arabidopsis \nseedlings pre-treated at 28°C or with benzyl alcohol (BA). Measurement by qPCR of \nthe fold increases in a) and c) ICS1 and b) and d) EDS1 transcript expression in \nArabidopsis seedlings pre-treated for 24 h at a) and b) 20°C or 28°C, or c) and d) at 20°C \nwith 0 mM (H2O) or 30 mM benzyl alcohol (BA), in response to water or 0.5 μM flg22 1, \n3, and 6 h after the start of treatment. Relative Quantification (RQ) values were calculated \nafter normalisation to PEX4 expression levels. The value produced for each treatment is \nthe mean of three biological replicates, with each biological replicate representing the \nmean value of three technical repeats. Error bars represent the S.E.M and significant \ndifferences (p ≤0.05) were determined using ANOVA and the Tukey HSD post hoc test at \na 95% confidence interval. Bars with the same letter are not significantly different.  \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 30 \n \n \n \nFigure 3. Basal FLS2 protein levels are lower in Arabidopsis seedlings pre -treated \nat 28°C or with benzyl alcohol (BA). Total protein was extracted from 14-day old pMAQ2 \nor fls2C (control) Arabidopsis seedlings pre-treated at a) 20°C or 28°C or b) at 20°C with \n0 mM or 30 mM benzyl alcohol (BA) for 24 h. FLS2 levels were detected by western \nblotting. After detection, the blots were stained with Coomassie brilliant blue (CBB) to \ndisplay loading. Values indicate the size (KDa) of the bands with the expected size of \nFLS2 indicated on the blot. Each sample loaded onto the blot is a biological replicate.  \n \npMAQ2 pMAQ2fls2C fls2C\n20ºC28ºC\nα-FLS2\n(a)\nCBB\npMAQ2\nBA\npMAQ2\nH2O\nfls2C\nBA\n(b)\n170 kDa\nα-FLS2170 kDa\nCBB\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 31 \n \n \n \n \n \n \n \n \n \nFigure 4. Basal FLS2 protein levels are restored in Arabidopsis seedlings pre -\ntreated at 28°C or with benzyl alcohol (BA) using the pMDC7 FLS2 construct. Total \nprotein was extracted from 14-day old pMAQ2pMDC7FLS2 or fls2C (control) Arabidopsis \nseedlings pre-treated for 24 h at 20°C, 28°C or with 30 mM benzyl alcohol (BA). Seedlings \nalso underwent a 16 h treatment of 10 μM estradiol (+) or 0.02% (v/v) DMSO ( -) before \nprotein extraction. FLS2 levels were detected by western blotting. After detection, the \nblots were stained with Coomassie brilliant blue (CBB) to display loading. Values indicate \nthe size (KDa) of the bands with the expected size of FLS2 indicated on the blot.  \n \n \nfls2C pMAQ2pMDC7FLS2\n+-+-+-+-\nα-FLS2\n20ºC 20ºC 28ºC BA\nCBB\n170 kDa\nEstradiol\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 32 \n \n \nFigure 5. Flg22 -induced [Ca2+]cyt increases are restored in Arabidopsis seedlings \npre-treated at 28°C or with benzyl alcohol (BA) using the pMDC7 FLS2 construct. \n[Ca2+]cyt changes in response to 0.5 μM flg22 in pMAQ2pMDC7 FLS2 Arabidopsis \nseedlings pre-treated for 24 h a) 28°C or b) at 20°C with 30 mM BA and concurrently pre-\ntreated for 16 h with 10 μM estradiol (EST) or 0.02% (v/v) DMSO. The traces shown are \nmeans of 15 replicate measurements and the error bars represent the S.E.M. The \naverage area under the  curve (AUC  (µM)) ± S.E.M (SE) values for the flg22 -induced \ncalcium responses (100-300 s) are shown. The AUC values are the means of 15 replicate \nresponses and the p value shown is the significance of the differences in the AUC as \ndetermined by a pairwise t-test.  \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 33 \nFigure 6. Flg22 -dependent ICS1 and EDS1 expression is restored in Arabidopsis \nseedlings pre-treated at 28°C or with benzyl alcohol (BA) using the pMDC7 FLS2 \nconstruct. Measurement by qPCR of the fold increases in a) ICS1 and b) EDS1 transcript \nexpression in pMAQ2pMDC7 FLS2 Arabidopsis seedlings pre -treated for 24 h at 20 °C, \n28°C or with 30 mM benzyl alcohol (BA) in response to water or 0.5 μM flg22 3 h after the \nstart of treatment. The seedlings were also concurrently pre -treated for 16 h with 10 μM \nestradiol or 0.02% (v/v) DMSO. Relative Quantification (RQ) values were calculated after \nnormalisation to PEX4 expression levels. The value produced for each treatment is the \nmean of three biological replicates, with each biological replicate representing the mean \nvalue of three technical repeats. Error bars represent the S.E.M and significant \ndifferences (p ≤0.05) were determined using ANOVA and the Tukey HSD post hoc test at \na 95% confidence interval. Bars with the same letter are not significantly different.  \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 34 \nSupplementary Table 1 . Synthetic gene block sequences used for cloning the \nFLS2pMDC7 construct. \nName Sequence (5’ to 3’) \nGENE BLOCK 1: \nFLS2: 1-835 \nTGTACAAAAAAGCAGGCTCCGCGGCCGCATAACAATGAAGTTACTCT\nCAAAGACCTTTTTGATATTAACTCTCACCTTCTTCTTCTTTGGCATTGC\nACTAGCGAAACAGAGCTTTGAACCAGAGATCGAAGCTTTGAAATCCT\nTCAAGAATGGTATTTCCAACGACCCTTTAGGAGTATTATCAGATTGGA\nCCATCATCGGTTCGTTACGACACTGTAATTGGACCGGAATCACCTGC\nGATAGTACCGGACATGTAGTCTCGGTTTCCTTGCTGGAGAAGCAACT\nTGAAGGTGTTCTGTCTCCAGCCATAGCGAATCTCACCTATCTCCAGG\nTTCTTGATCTCACTTCAAATAGTTTTACCGGCAAAATACCGGCTGAAA\nTAGGAAAGTTAACCGAGCTTAACCAGCTTATTCTGTACCTAAACTATT\nTCTCTGGTTCGATTCCTTCTGGAATCTGGGAGCTTAAGAATATTTTCT\nATCTTGATCTTAGAAATAATTTGTTGTCCGGTGATGTTCCTGAGGAAA\nTCTGCAAAACCAGTTCTTTGGTATTGATTGGGTTTGATTACAACAACT\nTAACCGGGAAAATACCAGAATGCTTAGGAGATTTGGTTCATCTCCAAA\nTGTTTGTAGCAGCTGGTAACCATTTAACTGGTTCGATTCCGGTATCAA\nTTGGTACTCTGGCTAATTTAACGGATTTAGACCTGAGTGGTAACCAGT\nTAACCGGAAAAATACCGAGAGATTTTGGAAATCTCTTGAACTTACAGT\nCTCTCGTTTTAACTGAAAACTTGTTGGAAGGAGATATACCAGCTGAGA\nTCGGAAACTGCTCGAGCTTGGTC \nGENE BLOCK 2: \nFLS2: 811-1932 \nGATCGGAAACTGCTCGAGCTTGGTCCAACTTGAGCTTTACGATAACC\nAGTTAACCGGGAAAATACCAGCTGAATTAGGGAATTTGGTTCAGCTG\nCAAGCACTCCGGATATACAAGAACAAACTTACTTCTTCAATTCCATCT\nTCATTGTTCCGGTTAACTCAGTTAACCCATTTGGGGTTATCAGAAAAC\nCATTTGGTTGGACCGATATCAGAAGAAATCGGTTTTCTTGAGTCACTT\nGAAGTCCTCACACTTCATTCCAACAACTTCACAGGAGAGTTTCCACAG\nTCCATCACAAACTTGAGGAACTTGACAGTCCTAACGGTGGGGTTCAA\nTAATATTTCCGGTGAGCTCCCGGCGGATCTAGGGCTTCTTACAAACC\nTTCGGAACCTTTCAGCGCACGACAATCTTCTTACCGGACCAATACCTT\nCCAGCATAAGTAACTGCACCGGTCTTAAACTCCTGGACCTGTCTCAC\nAACCAAATGACTGGCGAGATCCCGCGGGGTTTCGGAAGGATGAATC\nTTACGTTCATTTCTATTGGGAGGAATCATTTCACCGGTGAAATTCCAG\nATGATATCTTCAACTGTTCAAACTTGGAAACTCTTAGTGTGGCAGATA\nACAACTTAACAGGAACTCTCAAGCCATTAATTGGGAAGCTTCAAAAAC\nTCAGGATTTTGCAAGTTTCATATAACTCTCTCACTGGACCGATTCCTC\nGAGAAATCGGGAATCTGAAAGATTTGAATATCTTGTACCTTCACTCTA\nATGGTTTCACAGGGAGAATCCCGAGAGAGATGTCGAATCTCACTCTC\nCTCCAGGGGCTAAGGATGTATTCAAATGATCTTGAAGGTCCAATTCCT\nGAAGAAATGTTTGATATGAAGCTACTCTCAGTTCTTGATCTTTCCAAC\nAACAAATTCTCAGGTCAAATTCCTGCCTTGTTCTCCAAGCTTGAATCG\nCTTACCTACTTGAGTCTTCAAGGAAACAAATTCAACGGGTCTATCCCT\nGCAAGCCTTAAGTCGCTTTCGCTTCTCAACACATTCGATATCTCCGAC\n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 35 \n \n \n \n \n \nAATCTTCTCACTGGAACCATCCCTGGAGAGCTGTTAGCTTCTTTGAAA\nAACATGCAGCTTTACCTCAACTTCTC \nGENE BLOCK 3: \nFLS2: 1908-3584 \nACATGCAGCTTTACCTCAACTTCTCAAACAACTTGTTGACTGGAACCA\nTCCCAAAGGAGCTTGGAAAGCTTGAAATGGTTCAAGAAATCGACCTT\nTCAAACAATCTCTTTTCTGGGTCTATTCCAAGATCTTTACAGGCCTGC\nAAAAATGTGTTCACACTGGATTTTTCGCAGAACAATCTCTCGGGTCAT\nATACCAGATGAAGTCTTCCAAGGCATGGATATGATCATAAGCTTGAAC\nCTTTCAAGGAACAGTTTCTCTGGAGAAATCCCTCAGAGCTTCGGGAA\nCATGACGCATTTGGTCTCCTTGGATCTCTCTAGTAACAATCTCACTGG\nTGAAATTCCAGAGAGTCTCGCCAATCTTTCGACTCTGAAACATCTCAA\nACTAGCTTCAAACAACCTCAAAGGCCATGTTCCTGAATCCGGGGTGT\nTCAAAAACATCAACGCCTCTGATCTAATGGGAAACACAGATCTCTGTG\nGTAGCAAGAAGCCTCTCAAGCCATGTACGATCAAGCAGAAGTCGAGC\nCACTTCTCGAAGAGAACCAGAGTCATCCTGATTATTCTTGGATCAGCC\nGCGGCTCTTCTTCTTGTCCTGCTTCTTGTTCTGATTCTAACCTGTTGC\nAAGAAAAAAGAAAAAAAGATTGAAAATTCATCAGAGTCCTCATTACCG\nGATTTGGATTCAGCTCTGAAACTGAAGAGATTTGAACCAAAAGAGTTG\nGAGCAAGCAACAGATTCATTCAACAGTGCCAACATCATTGGCTCAAG\nCAGCTTAAGCACAGTGTACAAAGGTCAGCTAGAAGATGGGACAGTGA\nTTGCAGTAAAAGTATTGAATCTAAAGGAATTCTCTGCAGAATCAGACA\nAGTGGTTCTACACAGAAGCTAAAACATTGAGCCAACTAAAACATCGAA\nACCTGGTGAAGATCTTAGGGTTTGCGTGGGAAAGCGGCAAAACGAAA\nGCTTTAGTGCTTCCATTTATGGAGAATGGAAACTTGGAGGACACCATT\nCACGGCTCTGCAGCACCGATTGGGTCGCTTTTAGAAAAAATCGATCT\nTTGTGTTCATATCGCAAGCGGAATCGATTATCTTCATTCTGGATATGG\nTTTTCCCATCGTTCATTGTGATCTGAAGCCAGCTAATATACTCCTTGA\nCAGTGACCGCGTTGCTCACGTAAGCGATTTTGGAACTGCTCGGATTC\nTAGGTTTCCGCGAAGATGGAAGCACCACAGCTTCAACATCAGCCTTC\nGAGGGTACAATTGGATACTTAGCTCCAGAGTTTGCTTATATGAGGAAA\nGTGACAACAAAAGCCGATGTATTCAGCTTCGGGATCATAATGATGGA\nGCTGATGACGAAACAGAGACCAACTTCGTTGAATGATGAAGATTCAC\nAAGACATGACTTTGCGCCAATTGGTGGAGAAATCGATTGGAAATGGA\nAGAAAAGGGATGGTTAGGGTTCTTGATATGGAACTCGGGGACTCTAT\nTGTTTCTCTGAAACAGGAAGAGGCTATTGAAGACTTTCTGAAGCTTTG\nTTTGTTCTGTACAAGCTCTAGACCTGAAGATCGACCTGATATGAACGA\nGATTCTTACACATCTGATGAAACTTAGAGGCAAAGCGAATTCATTTCG\nAGAAGATCGTAACGAGGATCGAGAAGTTTAGGGCGCGCCGACCCAG\nCTTTCTTGTACAA \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint \n\n 36 \n \n \nSupplemental Figure 1. Confirmation of anti-FLS2 antibody specificity.  \nProteins samples from Col -0 or fec seedlings were separated in an 8% SDS -PAGE gel \nfollowed by transfer to PVDF and immunoblotting with anti -FLS2 antibodies. After \nimaging, the membrane was stained with Coomassie Brilliant Blue G250 to show loading. \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(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 \nThe copyright holder for this preprintthis version posted October 20, 2025. ; https://doi.org/10.1101/2025.10.20.683271doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}