# Source paper archive - Source title: Thermosensory pathway makes a U-turn to respond to heat - Author(s): Luciana Bianchimano, Cha San Koh, Maria Belen De Luca, Mauro German Murcia, Matias Ezequiel Pereyra, Anna B. Hamacher, Matias Zurbriggen, Jorge Casal - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.11.06.764838v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.11.06.764838v1.full.pdf - PDF SHA-256: d130f80a744f15310d6589cd8ee5503d032c831c4f730a31502941933e32c5e8 - Converter: pymupdf4llm 0.0.27 - Limits: Automated PDF text extraction; reading order, equations, tables and formatting may be incomplete or incorrect. Images are omitted, and no OCR was performed. Markup and links are neutralized for safe downloading. Consult the original PDF for the authoritative version. --- [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1 # Thermosensory pathway makes a U-turn to respond 2 to heat 3 4 ### 5 Luciana Bianchimano [1], Cha San Koh [2,6], María Belén De Luca [3,6,7], Germán 6 Murcia [1,6], Matías Ezequiel Pereyra [1,3], Anna B. Hamacher [4], Matias D. 7 Zurbriggen [2,5], Jorge J. Casal [1,3] 8 9 1 Fundación Instituto Leloir and IIBBA-CONICET. Av. Patricias Argentinas 435, Buenos Aires 10 C1405BWE, Argentina. 11 2 Institute of Synthetic Biology, University of Düsseldorf, 40225 Düsseldorf, Germany 12 3 Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas 13 (CONICET), Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura (IFEVA), 14 Facultad de Agronomía, Buenos Aires, Argentina. 15 4 Center for Advanced Imaging (CAi), University of Düsseldorf, 40225 Düsseldorf, Germany 16 5 CEPLAS-Cluster of Excellence in Plant Sciences, University of Düsseldorf, 40225 Düsseldorf, 17 Germany 18 6 These authors contributed equally to this work. 19 7 Current address: L'Institut des Sciences des Plantes, Universidad Paris-Saclay. 630 Rue Noetzlin 20 Bâtiment, 91190 Gif-sur-Yvette, Paris, France. 21 22 23 Author for correspondence: 24 Jorge J. Casal 25 Email: casal@agro.uba.ar 26 1 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) ## 27 Summary 28 29 Plant organs exhibit a unimodal growth response to temperature, yet the mechanistic links 30 connecting growth-promoting and growth-inhibiting thermal ranges remain unresolved. In 31 _Arabidopsis_ seedlings, warm temperatures promote hypocotyl elongation by relieving phytochrome 32 B (phyB) inhibition, thereby activating the transcription factors PIF4 and PIF7 to drive auxin 33 synthesis. Here, we demonstrate that temperatures beyond the growth optimum actively suppress 34 growth by downregulating auxin levels, auxin signalling, and PIF4 abundance specifically within 35 cotyledons, the primary site of auxin production. Strikingly, whereas warm temperatures induce the 36 shrinking of phyB nuclear bodies, heat stress causes phyB to assemble into enlarged condensates, 37 even when phyB is predominantly in its inactive form. We recapitulated heat-induced phyB 38 condensates in mammalian cells, demonstrating an intrinsic biophysical mechanism. Together, our 39 findings indicate that heat-induced growth inhibition is not merely passive stress damage, but an 40 active molecular reversal of the thermomorphogenic promotion pathway mediated by distinct phyB 41 condensates. 42 2 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) ## 43 Introduction 44 Growth of plant organs typically follows a unimodal curve, increasing with temperature to an 45 optimum and declining at higher temperatures (Parent & Tardieu, 2012; Zhu _et al._, 2022). The 46 optimum growth temperature is generally higher for species originating from tropical regions than 47 for those from temperate regions (Parent & Tardieu, 2012). Even within a species, organs differ in 48 their thermal responses. In _Arabidopsis thaliana_, hypocotyl elongation increases between 10°C and 49 30°C (Legris _et al._, 2017), primary root elongation increases between 10°C and 25°C (Ibañez _et al._, 50 2017; Yang _et al._, 2017; Borniego _et al._, 2022) whereas cotyledon expansion can decline between 51 20°C and 28°C (Hahm _et al._, 2020; Costigliolo Rojas _et al._, 2022). 52 Our understanding of the mechanisms involved in the promotion of hypocotyl growth by warm 53 temperatures has improved substantially during the last decades (Quint _et al._, 2023). Warm 54 temperatures enhance auxin synthesis in the cotyledons, followed by hormone transport to the 55 hypocotyl to drive elongation (Gray _et al._, 1998; Franklin _et al._, 2011; Bellstaedt _et al._, 2019). The 56 bHLH transcription factors PHYTOCHROME INTERACTING FACTOR 4 (PIF4) (Franklin _et al._, 2011; Sun 57 _et al._, 2012) and PIF7 (Fiorucci _et al._, 2020) activate auxin biosynthesis genes. PIF7 accumulates via 58 warm temperature-dependent translational regulation (Fiorucci _et al._, 2020; Chung _et al._, 2020), 59 while _PIF4_ increases via transcriptional (Koini _et al._, 2009) and post-translational (Foreman _et al._, 60 2011; Zhang _et al._, 2025; Tan _et al._, 2026) controls. Upstream, the EVENING COMPLEX (EC) represses 61 _PIF4_ expression at night (Nusinow _et al._, 2011), but one of its components, EARLY FLOWERING 3 62 (ELF3), undergoes temperature-induced phase separation into nuclear condensates that prevent 63 the EC formation (Jung _et al._, 2020; Silva _et al._, 2020). PIF5 and PIF3 further contribute to 64 thermomorphogenesis (Romero-Montepaone _et al._, 2021). 65 The photoreceptor phytochrome B (phyB) represses PIF4 accumulation and intrinsic ability to bind 66 to transcriptional targets (Park _et al._, 2018; Pham _et al._, 2018). Warm temperatures accelerate 67 thermal reversion of phyB from the biologically active far-red light (FR)-absorbing Pfr form to its 68 inactive red light (R) absorbing Pr form (Jung _et al._, 2016; Legris _et al._, 2016). phyB Pfr assembles 69 into nuclear condensates or nuclear bodies (NBs) through liquid-liquid-phase separation (Kircher _et_ 70 _al._, 1999; Yamaguchi _et al._, 1999; Chen _et al._, 2003, 2022). These NBs dissociate upon FR irradiation 71 (Rausenberger _et al._, 2010; Van Buskirk _et al._, 2014) and shrink at elevated temperatures (Legris _et_ 72 _al._, 2016; Hahm _et al._, 2020; Chen _et al._, 2022). phyB NBs are multi-protein condensates containing 73 both primary and secondary interacting partners (Kim _et al._, 2023). Although phyB is able to form 74 condensates when expressed in mammalian cells in the absence of other plant proteins (Chen _et al._, 75 2022), the size of these condensates increases substantially in the presence of interacting 76 partners(Beyer _et al._, 2015b; Chen _et al._, 2022). In plant cells, NBs formation also depends on several 77 such factors, including PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) and PCH1-LIKE (PCHL) 78 (Galvão _et al._ ; Enderle _et al._, 2017; Huang _et al._, 2019; Fang _et al._, 2022). 79 Although warm temperatures promote hypocotyl growth through well-understood mechanisms, 80 how heat represses growth remains elusive. This inhibition could reflect cellular damage (such as 81 membrane dysfunction, protein denaturation, or reactive oxygen species) or an actively regulated 82 pathway. One hypothesis is that heat further amplifies warm-temperature growth pathways, 83 causing auxin-induced apoplastic acidification to reach supra-optimal intensities known to inhibit 84 hypocotyl growth (Wang _et al._, 2025). Instead, we find that heat reverses key processes underlying 3 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 85 hypocotyl thermomorphogenesis. Because warm temperatures accelerate phyB thermal reversion 86 and promote NBs disassembly, phyB condensates would be expected to decline at higher 87 temperatures. Unexpectedly, quantitative imaging in plants, together with an orthogonal 88 reconstitution system in mammalian cells, reveals that heat enlarges phyB condensates, uncovering 89 a temperature-dependent reorganization of phyB associated with growth repression. 90 ## 91 Materials and methods 92 **Plant material** 93 94 We used _Arabidopsis thaliana_ seedlings of the genotypes listed in Table S1. Seeds were sown on 1 95 % agar-water in clear plastic boxes of 3 cm length, 4 cm width and 2 cm height, and stratified 4 d at 96 5 ºC in darkness. Then, the boxes were transferred to a growth room at 22°C with a photoperiod of 97 10 h of 80-90 μmolm [−2] s [−1] of white light (400–700 nm) provided by a mixture of fluorescent and 98 halogen lamps (red/far-red ratio = 1.1). In hypocotyl-growth experiments, the seedlings were grown 99 parallel to the agar surface, which was placed vertically. For other determinations, the seedlings 100 were grown with their hypocotyls perpendicular to the agar surface, which was placed horizontally. 101 For luminometer experiments, the seedlings were grown in 96-well plates containing 1 % agar102 water. One hour after the beginning of the fourth day (time= 0 h), the seedlings were transferred 103 to the different treatments. 104 105 **Temperature treatments** 106 107 Unless indicated otherwise, 1 h after the beginning of the fourth day (time= 0 h), the seedlings were 108 transferred to the different temperatures (10°C, 15°C, 22°C, 28°C, 33°C or 37°C), where they 109 remained 9 h (hypocotyl growth), 4h or 8h (other observations) without changes in light conditions. 110 111 In selected experiments, the seedlings were transferred at time= 0 h to different combinations of 112 temperature (28°C, 33°C or 37°C) and R pulses (5 min, 35 μmol m [-2] s [-1] ) or FR pulses (5 min, 15 μmol 113 m [-2] s [-1] ) followed by darkness, hourly R pulses or to continuous high-irradiance R (175 μmol m [-2] s [-1] ). 114 The R and FR treatments (wavelengths of maximum emission at 660 nm and 730 nm, respectively) 115 were provided by LEDs. The specific sequence of conditions is detailed along with the description of 116 the results. 117 118 **Rate of hypocotyl elongation** 119 For each biological replicate, 10, 12 or 6 seedlings (see figure legends) were photographed from the 120 side with a digital camera (PowerShot; Canon, Tokyo, Japan) at the beginning of treatments (time= 121 0 h) and at the end of the photoperiod (time= 9 h) of the fourth day. Hypocotyl length was 122 determined at both time points using image-processing software (Sellaro _et al._, 2009) and used to 123 calculate the increments over the 9-h period. 124 125 **Quantifying genetic contributions to hypocotyl growth** 126 127 Hypocotyl growth (G) was modeled as the ratio of maximum growth capacity (G max ) to the total 128 inhibitory activity (I) plus a constant of one, representing growth in the absence of inhibition: 4 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 129 130 𝐺= [G] [!"#] 1 + 𝐼 131 132 In this model, total inhibitory activity in the wild type is the sum of the contribution from gene X (I X ) 133 and other systemic components (I other ). In the gene _X_ mutant, inhibitory activity is attributed solely 134 to these other components. Therefore, I other was first calculated using the growth equation for the 135 mutant: 136 I $%&'( = [G] [!"#] ~~)~~ 𝐺 137 I $%&'( = [!"#] ~~)~~ 𝐺 - −1 𝐺 !)%"*% 138 139 This value was then substituted into the wild-type equation to isolate the specific inhibitory 140 contribution of gene _X_ : 141 142 I # = 𝐺 !"# −1 −𝐼 $%&'( 𝐺 ~~,~~ +,-. %01' ~~-~~ 143 144 Because _pifs_ and _bes1/bzr1_ mutants exhibit reduced growth relative to the wild type, their 145 calculated inhibitory contributions yielded negative values. To reflect their biological roles as growth 146 enhancers, these effects are presented as promotion, yielding positive values. Maximum growth 147 capacity (G max ) was defined using _phyB_ mutant data from plants grown under shade at 28°C. 148 149 **Confocal microscopy** 150 151 Confocal images from Arabidopsis seedlings were taken from live samples except in the case of the 152 experiments where light pulses were followed by darkness to analyse phyB-GFP NBs. In the latter 153 cases, to avoid any spurious exposure to light that could distort the results, the seedlings were fixed 154 by immersion in 1% paraformaldehyde in 1× PBS, applying a vacuum for 24 seconds, followed by 155 two 30-minute washes with NH 4 Cl and suspension in 1× PBS, as described (Pereyra _et al._, 2023). In 156 all cases, images were taken of the epidermis or the first sub-epidermal layers, taken 4 h and 8 h 157 after the application of treatments of temperature. The organs observed (cotyledons or hypocotyls) 158 are indicated in each case. 159 160 For the analysis of the _AuxSen_ (Herud-Sikimić _et al._, 2021) and Apo-pHusion (Gjetting _et al._, 2012) 161 reporters, confocal fluorescence images were taken with the LSM 510 Meta (Zeiss) laser scanning 162 confocal microscope with an Plan-Apochromat 40×/1.2 objective lens. For FRET ratio measurement 163 of _AuxSen_, the donor fluorophore was excited with a 405 nm wavelength laser bean, and the 164 emission of the donor was detected between 460 nm and 500 nm, while the emission of the 165 acceptor (FRET) was detected between 505 nm and 550 nm. Additionally, to verify its performance, 166 the acceptor was excited with a 488 nm wavelength laser, and its emission was detected between 167 505 nm and 550 nm. Data correspond to the ratio between the emission intensities of the acceptor 168 (505-550 nm) and donor (460-500 nm). For Apo-pHusion, we used a sequential scanning of EGFP 169 and mRFP1 proteins. EGFP was excited at 488 nm and detected between 500 nm and 550 nm while 170 mRFP1 was excited at 585 nm and detected between 600 nm and 630 nm, as described (Gjetting _et_ 171 _al._, 2012). Data correspond to the ratio between EGFP and mRFP1 fluorescence intensities. 5 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 172 To quantify nuclear fluorescence, a Zeiss LSM 710 NLO confocal microscope was used, which allows 173 the separation of the signal emitted by fluorescent proteins from that of chloroplasts. For 174 visualisation of proteins fused to GFP and YFP, mCitrine, and DII-VENUS, we used an Argon excitation 175 laser (λ = 488 nm), and fluorescence was detected using a BP 505-530, 505-550, and 505-570 filter, 176 respectively. Since chloroplasts exhibit strong autofluorescence, and chlorophylls are excited and 177 fluoresce in wavelength ranges within those used for the excitation and detection of fluorescent 178 proteins, a separate channel with a He-Ne excitation laser (λ = 543 nm) and an LP 630 filter was used 179 to detect chlorophyll and distinguish chloroplasts in the fluorescent protein channel. 180 181 Nuclear fluorescence is expressed per cell as the ratio between the integral of nuclear fluorescence 182 and the number of cells within the area under analysis. Their spindle shape, compressed against the 183 cell wall, identified nuclei and in many cases, the presence of the nucleolus was detected. The 184 average fluorescence intensity within the nuclei and the number of nuclei within the area under 185 analysis were recorded by using Fiji software (Schindelin _et al._, 2012), and their product defined the 186 integral of nuclear fluorescence within the area under analysis. The area per cell was measured in 187 5-10 cells and the total area under analysis were measured with the aid of the transmission and 188 chlorophyll channels, and these data were used to calculate the number of cells within the area 189 under analysis. 190 191 To analyse phyB NBs, we used LSM 510 Meta (Zeiss) to first obtained the image of the area to be 192 analysed, and then performed 8× magnifications of that image in the areas of interest focused on 193 one nucleus to optimise the visualisation of NBs. The area, number and fluorescence of the NBs 194 were obtained by using an automatic analysis tool developed in Icy (de Chaumont _et al._, 2012; Legris 195 _et al._, 2016) by using Fiji software (Schindelin _et al._, 2012). 196 197 **Quantitative RT-PCR** 198 199 For each biological replicate, approximately 50 mg of Col-0 seedlings were harvested, frozen in liquid 200 nitrogen, and ground with a mortar and pestle to a fine powder, 4 h after the beginning of the 201 temperature treatments. Total RNA was extracted using Trizol reagent (Invitrogen) according to the 202 manufacturer's instructions. One μg of RNA was treated with RQ1 RNase-Free DNase (Promega) and 203 reverse transcribed using Superscript II Reverse Transcriptase (SSII RT) (Thermo Fisher Scientific) 204 and oligo-dT, according to the manufacturer's protocol. The resulting cDNAs were then amplified 205 using an Mx3000P Real Time Cycler PCR System (Agilent Technologies). The _UBQ2_ gene transcript 206 (AT2G36170) was used as a reference. Quantitative RT-PCR analysis was performed using the 207 standard curve method (Agilent Technologies, 2012). Primers are listed in Table S1. 208 209 **Colorimetric auxin quantification** 210 211 Auxin levels were quantified following a colorimetric method (Manna _et al._, 2024). For each 212 biological replicate, 50-100 mg fresh weight of Col-0 seedlings were harvested 4 h after the onset of 213 temperature treatments, weighed, and flash-frozen in liquid nitrogen. Samples were ground to a 214 fine powder using a mortar and pestle and extracted in 1 mL of absolute ethanol. The mixture was 215 swirled to ensure IAA solubilisation and incubated on ice for 10 min, followed by centrifugation at 216 1,000× _g_ for 10 min at 4 °C. For the colorimetric reaction, 80 µL of the resulting supernatant was 217 combined with 100 µL of Salkowski reagent (distilled H 2 O, concentrated H 2 SO 4, and 0.5 M FeCl 3 in a 218 25:15:0.75 ratio) in a 96-well transparent microplate. Following a 30-min incubation at room 219 temperature in the dark, absorbance was measured at 540 nm using a Varioskan LUX multimode 6 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 220 reader (Thermo Fisher Scientific). IAA concentrations were determined using a standard calibration 221 curve (range: 0.49–501.76 mg/L, Table S1) and normalised to the fresh weight of each sample. 222 223 **Luminometer readings** 224 225 Transgenic seedlings bearing the LUC reporter were incubated with 30 μL of 0.5 mM D-luciferin per 226 well 24 h before starting to record luciferase activity as described (Murcia _et al._, 2022). Luciferase 227 (LUC) activity was recorded with a Varioskan™ LUX multimode microplate reader (Thermo Fisher 228 Scientific) for three seconds, 4 h after the beginning of the temperature treatments. 229 230 **Protein blots** 231 232 For each biological replicate, approximately 50 mg of seedlings were harvested and frozen in liquid 233 nitrogen and ground with a mortar and pestle to a fine powder, 4 h or 8 h after the beginning of the 234 temperature treatments. Blot reactives are listed in Table S1. Total protein was extracted on a buffer 235 containing 125 mM Tris-HCl pH 7.4, 0.2% SDS, 10% glycerol, 6 M urea, and 1% β236 mercaptoethanol(Nieto _et al._, 2015). Samples were heated for 3 min at 95°C, then centrifuged for 237 15 min at 13,000 rpm, and the supernatant was removed. Proteins were separated by PAGE using 238 12% (phyB, PIF7) or 8% (PIF4) gels. Gels were then electrotransferred onto a 0.45 µm nitrocellulose 239 membrane (Thermo Fisher catalogue 88018) using a wet transfer system for 2 h at 100 V. Rabbit 240 polyclonal anti-HA and mouse polyclonal anti-GFP (Thermo Fisher catalogue PA1985 and A-11122) 241 were used as primary antibodies. Anti-rabbit IgG (H+L) with HRP (Thermo Fisher catalogue 32460) 242 and anti-mouse (H+L) were used as secondary antibodies. Detection was carried out using the 243 SuperSignal [TM ] West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific catalogue 244 34580) and X-ray films. Ponceau red staining was used as a control for loading uniformity. Band 245 quantification was carried out using the Fiji program, always selecting the same rectangle size. Full 246 blots corresponding to each biological replicate are shown in Figures S1, S2 and S3. The intensity of 247 each protein band was divided by the intensity of the Ponceau band to calculate its relative 248 abundance (see Dataset 1 for the data corresponding to the quantification and normalisation of 249 each band). 250 251 **β-Glucuronidase (GUS) activity measurements** 252 253 For each biological replicate, approximately 20 transgenic seedlings carrying the _pDR5:GUS_ reporter 254 were harvested 4 h after the start of temperature treatments. Samples were ground at ambient 255 temperature in 200 µL of extraction buffer (250 mM phosphate buffer pH 7, 0.5 M Na 2 EDTA, pH 8, 256 10% SDS, 10% Triton X-100, and 14.4 M β-mercaptoethanol) and immediately placed on ice. Extracts 257 were clarified by centrifugation at 12,000 rpm for 5 min (4 °C). To measure activity, 5 µL of 258 supernatant was combined with 195 µL of reaction buffer (extraction buffer supplemented with 25 259 mM 4-methylumbelliferyl β-D-glucuronide [4-MUG]) in a 96-well microtiter plate. The plates were 260 incubated at 37 °C for 1.5 h in darkness before the reaction was terminated using a carbonate stop 261 buffer (Na 2 CO 3 and NaOH). Fluorescence was determined at an excitation wavelength of 365 nm 262 and an emission wavelength of 455 nm using a Varioskan LUX reader (Thermo Fisher Scientific) 263 (Blázquez, 2007). Activity was quantified using a standard curve of 4-methyl umbelliferone (4-MU) 264 and normalised to the number of seedlings. 265 266 **Conventional confocal microscopy of mammalian cells** 267 7 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 268 Chinese hamster ovary cells (CHO-K1) were co-transfected with the _pSV40:PHYB-eGFP_ and 269 _pSV40:PIF3-mCerulean,_ _pSV40:PIF4-mCerulean_ or _pSV40:PIF7-mCerulean_ plasmids (cell and 270 recombinant materials are listed in Table S1). The plasmids were constructed using Aqua 271 cloning(Beyer _et al._, 2015a) and documented using GMOCU (Wagner _et al._, 2024). CHO-K1 cells 272 were cultured in Ham's F12 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% 273 (v/v) penicillin/streptomycin. Twenty-four hours prior to transfection, 50,000 cells per well were 274 seeded onto coverslips in 24-well plates in 500 μl of medium. For transfection, 0.75 μg of DNA was 275 diluted in 50 μl of Opti-MEM and combined with a PEI/Opti-MEM mixture [2.5 μl of PEI (1 mg/ml) in 276 50 μl of Opti-MEM per well] (Blomeier _et al._, 2021). Following a brief incubation, 100 μl of the 277 transfection complex was added dropwise to each well. The medium was replaced after 4 h to 278 minimise cytotoxicity. Twenty-four hours post-transfection, cells were incubated with 15 μM PCB 279 chromophore (Beyer _et al._, 2015b) for 1 h before applying the light and temperature treatments 280 specified in the figure legends. 281 282 The cells were fixed in the dark using 4% PFA (10 min on ice, followed by 10 min at room 283 temperature), with PBS washes performed before and after fixation. Nuclei were stained with 0.1% 284 DAPI for 5 min at room temperature. Finally, coverslips were mounted onto microscope slides using 285 8 μl of Mowiol/DABCO. The images were analysed as described for plant samples. 286 287 **High-content imaging of transfected mammalian cells** 288 289 The three constructs used for high-content imaging experiments using CHO-K1 cell line are 290 _pSV40::PIF3-eGFP::tSV40_ (pMZ725) [33], _pSV40::PHYB-mCherry::tSV40_ (pSJ025) [33] and 291 _pSV40::PHYB_ _[Y276H]_ _-mCherry::tSV40_ (pCSK208A). 292 293 CHO-K1 cells were initially maintained in Ham’s F12 medium (PAN Biotech, Aidenbach, Germany; 294 P04–14500) supplemented with 10% (v/v) tetracycline-free FBS and 1% (v/v) penicillin/streptomycin 295 (PAN Biotech). They were then gradually adapted over 2 weeks to 75% CO₂-independent medium 296 (Gibco #18045054) supplemented with GlutaMAX™ (Gibco #35050061), 10% FBS, 1% 297 penicillin/streptomycin, tetracycline and 25% Ham’s F12 to support CHO-K1 growth. This CO₂298 independent formulation, lacking HEPES, maintains physiological pH without external CO₂. 299 Experiments were performed at 15°C, 22°C, 28°C, and 37°C. Adapted CHO-K1 cells (about 14,000 300 cells in 150 μl medium per well) were seeded into PhenoPlate™ 96-well microplates (Revvity) and 301 incubated for 1 day at 37°C with 5% CO₂. For transfection in 96-well format, 0.2 μg total DNA per 302 well was diluted in Opti-MEM™ with P3000™ reagent (2 μl μg [-1] DNA) to a final volume of 5 μl. The 303 diluted DNA was mixed 1:1 with diluted Lipofectamine™ 3000 in Opti-MEM™ to form a 10 μl DNA– 304 lipid complex, incubated for 10–15 min at room temperature, and added to cells. Transfected cells 305 were incubated for 24 h at 37°C with 5% CO₂. Although removal of transfection complexes is not 306 required, a medium exchange 4–5 h post-transfection improved imaging quality by reducing 307 background. The following day, 15 μM phycocyanobilin (PCB; Frontier Scientific, P14137-50mg) was 308 added and cells were incubated for 1 h at 37°C in darkness. Plates were then illuminated with red 309 light (660 nm peak, 20 μmol·m⁻²·s⁻¹) for 15 min at the indicated temperatures (15°C, 22°C, 28°C, 310 37°C) in temperature-controlled chambers. Under continuous red light, the medium was rapidly 311 exchanged with chromophore-free medium pre-equilibrated to the respective temperature. Cells 312 were then incubated in complete darkness at the same temperature for 60 min. All subsequent 313 steps were performed under dim green light to mimic dark conditions. Cells were washed once with 314 100 μl pre-equilibrated PBS, then fixed in 100 μl 4% paraformaldehyde (PFA, Roth) for 15 min on 315 ice, protected from light. After removing PFA, cells were washed once with 100 μl PBS at room 8 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 316 temperature. To enable multiplexing in high-content analysis, two nuclear stains were used. Cells 317 were first incubated with 50 μl HCS NuclearMask™ Deep Red (Thermo Fisher H10294) for 45 min at 318 room temperature, protected from light. After one PBS wash, nuclei were counterstained with 50 319 μl DAPI (2 μg/ml) for 15 min. After removal of DAPI, cells were overlaid with 100 μl PBS and stored 320 at 4°C until imaging. 321 322 To ensure quantitatively unbiased and reproducible observations, a fully automated high-content 323 imaging workflow was implemented for fixed CHO-K1 cells. Imaging was performed on an Operetta 324 CLS High Content Imaging System (Revvity) at the Center for Advanced Imaging, Heinrich Heine 325 University Düsseldorf, equipped with a 4.7 MP sCMOS sensor (resolution of 2160 × 2160 pixels). 326 Excitation/emission settings were configured as follows: DAPI (355–385 nm / 430–500 nm; Nuclear 327 Counterstain), eGFP (460–490 nm / 500–550 nm; PIF3 condensates), mCherry (530–560 nm / 570– 328 650 nm; phyB condensates), and HCS NuclearMask Deep Red (615–645 nm / 665–760 nm). 329 330 Data acquisition was accelerated using the PreciScan module in Harmony™. An initial low331 magnification pre-scan (20× air objective, NA 0.4, binning 3) was used to identify cells containing 332 condensates, followed by automated image analysis using an optimised Harmony™ pipeline (Fig. 333 S4). High resolution imaging at 40x was performed using a custom Harmony™ analysis sequence 334 that integrated automated cell selection, stage repositioning, autofocus, and confocal z-stack 335 acquisition based on candidates identified during the pre-scan. 336 337 Confocal z-stacks were acquired using a 40× air objective (NA 0.75, binning 1), centred on the 338 identified cells. Per well, 100 – 200 fields were imaged depending on transfection efficiency. Z-stacks 339 comprised five optical sections for cells transfected with two plasmids and four optical sections for 340 cells transfected with a single plasmid, covering a z-range of 1.2-4.2 µm across the evaluated cells. 341 Non-transfected cells, defined by reporter fluorescence below a predefined threshold, were 342 automatically excluded. Nuclear and cytoplasmic segmentation were performed using NuclearMask 343 Deep Red and DAPI signals, respectively, while condensate detection was based on the fluorescence 344 channels described above. 345 346 Images were processed as maximum intensity projection (MIP) for condensate quantification using 347 a custom pipeline in Harmony™ version 5.2. The complete customised sequence analysis pipelines 348 are available from the authors upon reasonable request. 349 350 **KNIME-based data processing workflow** 351 352 Data analysis was performed using a customized workflow (Fig. S4) in KNIME Analytics Platform 353 (v5.11.0; www.knime.com), integrating Harmony™ image analysis outputs for segmentation and 354 feature extraction. Condensates were deconvoluted based on fluorescence properties. Cells in 355 contact with the image borders were excluded from the image analysis. Quality control was 356 performed in KNIME by applying cell-size thresholds to exclude artefacts and debris, as well as 357 filtering based on condensate signal intensity relative to the background. Condensates were 358 subsequently classified by subcellular localization using features derived from DAPI and 359 NuclearMask staining. A metadata harmonization step followed, whereby cell-level information was 360 systematically assigned to condensate (spot) data, enabling annotation at both single-cell and well 361 levels. Per-cell features, including condensate count, mean condensate intensity and condensate 362 size distribution, were computed from stringently filtered metadata restricted to double363 transfected cells (e.g., PhyB-PIF3). Final outputs were aggregated at the cell, well and condition 9 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 364 levels and exported as Excel files for downstream statistical analysis and visualization. All 365 experiments were performed in three independent replicates. An overview of the KNIME workflows 366 is provided here (Fig. S4), while the full workflows, including the details of the consolidated nodes, 367 are available from the authors upon reasonable request. 368 369 **Statistical analysis** 370 371 Statistical analyses were performed using GraphPad Prism (version 10). For experiments evaluating 372 temperature responses, data were fitted with linear or quadratic regression models. When a single 373 model failed to adequately describe the entire temperature range, separate regression lines were 374 fitted for the cold-to-warm and warm-to-hot intervals. The significance of the quadratic regression 375 was evaluated against the linear model, and the linear regression was tested against the null (mean376 only) model. 377 378 For comparisons involving only two temperature conditions, an unpaired Student’s _t_ -test was used. 379 For experiments involving three temperature conditions or factorial combinations of temperature 380 and other treatments (e.g., Picloram, R/FR pulses), one-way or two-way ANOVAs were employed, 381 followed by Tukey’s post-hoc tests for multiple comparisons. 382 383 Representative images were selected from the dataset used for quantitative analysis and are 384 displayed side-by-side. The quantitative values of these specific images reflect the mean values for 385 their respective conditions. For confocal microscopy, the observed fluorescence is specific to the 386 indicated probe, as all other signals were filtered out. 387 The meta-analysis of the expression of genes corresponding to the gene ontology term “response 388 to auxin” (GO:0009733) was conducted using the same procedures and list of published experiments 389 described earlier (Bianchimano _et al._, 2023). ## 390 Results 391 **Impaired growth inhibition by hot temperatures in auxin-related mutants** . 392 We cultivated seedlings of _Arabidopsis thaliana_ under short days at 22°C, transferred them to 393 temperatures ranging from 10°C to 37°C 1 h after the beginning of the fourth photoperiod and 394 measured hypocotyl elongation over the next 9 h. Wild-type Col-0 showed a monophasic growth 395 curve peaking at 28°C (Fig. 1a-b). Mutants impaired in auxin biosynthesis, the _shade avoidance 3_ 396 _(sav3)_ and in the _yucca3 (yuc3) yuc5 yuc7 yuc8 yuc9_ quintuple ( _yucq_ ), showed reduced elongation 397 at warm temperatures [14,21] and, strikingly, lacked the growth reduction between 28°C and 37°C (Fig. 398 1b). In these mutants, growth at cold temperatures was substantially lower than under heat, 399 demonstrating that inhibition was feasible but not observed. 400 Auxin promotes the expression of _SMALL AUXIN UP RNA (SAUR)_ genes, which help acidify the 401 apoplast to promote cell growth (Spartz _et al._, 2014; Weijers & Wagner, 2016; Vanneste _et al._, 2025). 402 The _saur_ multiple mutants ( _saur19_ - _24_ and _saur60-68, 75_ ) also failed to show growth inhibition by 403 heat compared to warmth (Fig. 1b), supporting the idea that growth inhibition at higher 404 temperatures may result from insufficient, rather than excessive, auxin. Consistently with this 405 interpretation, exogenous auxin partially restored hypocotyl elongation at 33°C (Fig. 1c). However, 10 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 406 exogenous auxin did not restore growth at 37°C, suggesting that additional auxin level-independent 407 mechanisms could contribute to heat-induced growth inhibition. 408 **Hot temperatures reduce auxin abundance and signalling** 409 Prompted by the impaired growth responses to heat observed in the auxin-related mutants, we 410 investigated the status of the canonical nuclear auxin signalling pathway. The levels of the hormone 411 measured in hypocotyl cells with the AuxSen reporter and by a colorimetric method in entire 412 seedlings peaked at 28°C and declined towards 33-37°C (Fig. 2a-b). Auxin signalling involves 413 decreased Aux/IAA co-receptors abundance and increased ARFs transcriptional activity, promoting 414 auxin-responsive gene expression (Weijers & Wagner, 2016; Vanneste _et al._, 2025). Consistently, 415 DII-VENUS reporters indicated minimal Aux/IAA stability at 28°C and higher levels at 33-37°C (Fig. 416 2c). The _DR5_ promoter is a reporter of the transcriptional activity of ARFs and peaked at 28°C, 417 declining at hot temperatures (Fig. 2d-f). 418 Using a meta-analysis approach previously developed in our group (Bianchimano _et al._, 2023), 419 temperature-responsive genes annotated with “response to auxin” similarly peaked at warm 420 temperatures and declined at hot temperatures (Fig. 2g). The canonical auxin pathway is active at 421 warm temperatures but declines under heat. 422 Auxin receptor TIR1-mCITRINE fluorescence in hypocotyls increased at 28°C (Wang _et al._, 2016) and 423 remained high at 33-37°C (Fig. 2h). In contrast, nuclear ARF6-GFP levels rose at 28°C (Wilkinson _et_ 424 _al._, 2026) but decreased under heat (Fig. 2i), supporting the above interpretation that signalling 425 components downstream the hormone itself are affected by high temperatures. 426 Heat-induced growth inhibition was also accompanied by apoplastic alkalization, as indicated by 427 increased fluorescence of the apoplast-targeted pH sensor Apo-pHluorin, which reports 428 extracellular pH with higher fluorescence meaning more alkaline conditions (Fig. 2j). This change is 429 consistent with reduced auxin signalling and opposite to the alternative hypothesis involving 430 excessive auxin, which inhibits growth by causing supra-optimal acidification (Wang _et al._, 2025). 431 **Functional analysis of key thermomorphogenic genes indicates a reversal of their physiological** 432 **impact at supra-optimal temperatures** 433 We measured hypocotyl growth in wild-type plants and in mutants of phyB and the PIFs (PIF4, PIF7, 434 PIF5, and PIF3, Fig. 3a). We then used these data to model the contribution of each gene to 435 hypocotyl growth inhibition or promotion under various temperature conditions (see the Methods 436 section for a detailed description of the calculations). As expected, comparing cold and warm 437 temperatures, the inhibitory contribution of phyB decreased, while the promotive contributions of 438 the PIFs increased (Fig. 3b). Surprisingly, this trend reversed at supra-optimal temperatures. The 439 inhibitory activity of phyB was enhanced, whereas the growth-promotion activities of PIF7, PIF4, 440 PIF5 and PIF3 declined (Fig 3b). 441 The transcription factor BRI1-EMS-SUPPRESSOR 1 (BES1) promotes hypocotyl growth at warm 442 temperatures by acting directly in the hypocotyl, rather than by influencing auxin synthesis in the 443 cotyledons (Ibañez _et al._, 2018; Costigliolo Rojas _et al._, 2022). Our data confirmed this function, 444 showing an increased promotive contribution between cold and warm temperatures (Fig. 3b). 11 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 445 Notably, BES1 did not show a reversal under heat (Fig. 3b), which aligns with its action outside the 446 main auxin-synthesis organ. 447 The transcription factor ELONGATED HYPOCOTYL 5 (HY5) inhibits hypocotyl elongation. The 448 observation that _hy5_ mutants exhibit hypersensitive hypocotyl growth at warm temperatures 449 (Delker _et al._, 2014) suggests that the inhibitory activity of HY5 is enhanced at warmer temperatures. 450 Our data supports this by showing maximal HY5-mediated inhibition at warm temperatures (Fig. 451 3b). Importantly, the inhibitory impact of HY5 decreased under heat, mirroring the reduced 452 promotive effect of PIF4 (Fig. 3b), is consistent with the established model that HY5 negatively 453 regulates PIF4 (Johansson _et al._, 2014; Gangappa _et al._, 2017). 454 **Reduced PIF4 protein abundance in the cotyledons at hot temperatures** 455 Motivated by the results of the genetic analysis, we investigated the abundance of PIF4 which is 456 known to promote auxin synthesis in cotyledons in response to warmth (Franklin _et al._, 2011; Sun 457 _et al._, 2012), The results revealed a complex, organ-specific response. 458 Using confocal microscopy, we observed that PIF4-GFP protein levels in cotyledons and hypocotyls 459 behaved differently. While the fluorescent signal increased in both organs when the temperature 460 shifted from cold to warm, it showed a distinct pattern at hot temperatures. In hypocotyls, PIF4-GFP 461 continued to rise, whereas in cotyledons, protein levels decreased (Fig. 4a-d). This reduction in 462 cotyledons was more pronounced after 8 h than at 4 h, suggesting a delayed inhibitory effect of high 463 temperature. 464 Additional experiments using protein blots and luminometer readings supported these findings. 465 Protein blots of whole seedlings, dominated by cotyledon tissue, showed maximal PIF4 abundance 466 at warm temperatures and, decreased at both temperature extremes, aligning with the cotyledon467 specific response (Fig. 4e, Fig. S1). Similarly, luminometer readings from PIF4-LUC, which are also 468 primarily influenced by the cotyledon signal, confirmed a decrease in PIF4 at hot temperatures (Fig. 469 4f). 470 **The different behaviour of PIF7, BES1, and HY5** 471 PIF7 is also involved in the promotion of auxin synthesis (Fiorucci _et al._, 2020). However, in contrast 472 to PIF4, its protein levels peaked at the highest temperatures tested in this study (Fig. 4g, Fig. S2), 473 likely driven by enhanced gene expression (Fig. 4h). Similarly, the nuclear abundance of BES1-GFP 474 continued to increase at supra-optimal temperatures (Fig. 4i). Conversely, HY5-YFP levels decreased 475 at warm temperatures, consistent with its known destabilisation (Park _et al._, 2017), but surprisingly 476 recovered under heat stress, exhibiting a response with a minimum signal at intermediate 477 temperatures (Fig. 4j). 478 While BES1 levels correlated with its physiological activity, the patterns of PIF7 and HY5 did not align 479 with their respective physiological impacts (Fig. 3b). This discrepancy suggests that the activities of 480 PIF7 and HY5 are likely modulated by complex protein-protein interactions or systemic components, 481 rather than being driven solely by their own temperature-dependent protein abundances. 482 **Increased phyB NB size at hot temperatures** 12 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 483 Previous studies indicate that phyB NB size peaks at mild temperatures and decrease at both cold 484 and warm extremes (Legris _et al._, 2016; Murcia _et al._, 2020). Using various phyB-GFP transgenic 485 lines, we confirmed this general trend in hypocotyl cells within the cold-to-warm range; however, 486 we also observed an unexpected increase in NB size at hot temperatures (Fig. 5a-c). This increase at 487 hot temperatures mirrors the physiological activity of phyB estimated from our genetic experiments 488 (Fig. 3b). Notably, these larger heat-responsive NBs were also present in cotyledon cells (Fig. S5). 489 In addition, phyB-GFP nuclear fluorescence increased across all tested temperatures (Fig. 5d-f), a 490 trend confirmed by protein blots, which showed a corresponding increase in total phyB protein 491 levels (Fig. 5g-h, Fig. S3 see also reference (Hahm _et al._, 2020)). While the decrease in phyB NB size 492 at higher temperatures is known to be caused by thermal reversion of phyB (Legris _et al._, 2016), the 493 increase in their size under hot conditions could be related at least partially to higher levels of 494 nuclear phyB (Ruff _et al._, 2018; Alberti _et al._, 2019; Villegas _et al._, 2022). 495 **High temperatures promote phyB NB formation despite FR-induced inactivation** 496 Since heat promotes the formation of large NBs while FR triggers their disassembly (Rausenberger 497 _et al._, 2010; Van Buskirk _et al._, 2014), we investigated the outcome of combining these antagonistic 498 stimuli. Arabidopsis seedlings were grown at 22°C and, 1 h after the beginning of the fourth 499 photoperiod, exposed to a 10-minute pulse of either R or FR before transfer to darkness at either 500 28°C or 37°C. 501 Seedlings transferred to 28°C for 4 h following an FR pulse exhibited only very small NBs (Fig. 6a). A 502 similar result was observed in seedlings receiving an R pulse. Although R initially establishes a high 503 proportion of the biologically active Pfr form, rapid thermal reversion at 28°C (Legris _et al._, 2016; 504 Burgie _et al._, 2021) gradually depletes this pool, leading to a progressive reduction in NB size(Van 505 Buskirk _et al._, 2014). Supporting this interpretation, NBs persisted at 28°C when R pulses were 506 applied hourly to maintain the active Pfr pool. 507 By contrast, when seedlings were transferred to 37°C, large NBs continued to be present even after 508 an FR pulse (Fig. 6a). This finding is remarkable because the FR pulse converts phyB to its inactive Pr 509 form, which normally promotes NB dissociation (Rausenberger _et al._, 2010; Van Buskirk _et al._, 2014). 510 These results suggest that extreme heat (37°C) exerts a potent effect on phyB NB stability that can 511 bypass the requirement for high levels of active Pfr, although the prior light treatment still 512 influenced the extent of assembly. This effect was specific to extreme heat, as it was observed at 513 37°C but not at 33°C (Fig. 6b). 514 To determine whether this thermal effect is reversible, seedlings incubated at 37°C for 4 h following 515 an FR pulse were transferred to 28°C for 1 h. Under these conditions, the NBs formed at 37°C 516 significantly decreased in size (Fig. 6c), indicating that the maintenance of these structures requires 517 sustained high temperature. 518 Next, we asked whether heat could trigger de novo NB formation. Seedlings treated with a FR pulse 519 were first kept at 28°C for 1 h to allow for near-complete NB disassembly (Fig. 6c). Remarkably, 520 when these seedlings were subsequently transferred to 37°C for 4 h, large phyB NBs successfully 521 reformed (Fig. 6c). These results show that 37°C promote NB assembly even after FR by enhancing 522 phyB nuclear levels and/or phyB propensity to condensate. 13 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 523 We further examined the genetic requirements for this process. In _pch1 pchl_ mutant seedlings 524 (Enderle _et al._, 2017; Huang _et al._, 2019), which strongly impair NB formation, FR treatment 525 followed by darkness yielded NBs approximately ten-fold smaller than in the wild type (Fig. 6d). 526 However, transfer to 37°C significantly increased NB size even in this background (Fig. 6d), further 527 emphasising the potency of the heat-induced assembly mechanism. 528 Finally, we assessed the physiological relevance of these observations by quantifying hypocotyl 529 growth in wild-type and _phyB_ mutant seedlings following a FR pulse and subsequent dark 530 incubation. Using the same approach of Figure 3B, we calculated phyB-dependent growth inhibition. 531 These measurements revealed a clear correlation between heat-induced NB enlargement and 532 enhanced phyB activity (Fig. 6e), suggesting that these assemblies are associated with a functional 533 signalling output. Although phyB activity is typically expected to be low following FR treatment, 534 phyB signalling can be amplified under conditions where only a small Pfr pool remains. For instance, 535 blue light is known to significantly enhance the plant responsiveness to the residual Pfr fraction 536 under FR conditions (Casal & Mazzella, 1998). 537 **High temperatures promote phyB NB enlargement in an orthogonal mammalian system** 538 To determine whether heat-induced phyB NB formation is dependent on the plant-cell molecular 539 environment, we expressed phyB and its interactor PIF3 in CHO-K1 cells supplemented with a 540 phycocyanobilin chromophore. Following a 15-minute R pulse, cells were incubated in the dark at 541 22°C, 28°C, or 37°C for 45 min prior to fixation. Consistent with thermal reversion effects, NB size 542 decreased between 22°C and 28°C. Strikingly however, at 37°C, cells exhibited significantly larger 543 NBs (Fig. 7a), resembling the heat-induced NB enlargement observed in Arabidopsis and revealing 544 a biphasic temperature response in which condensates shrink between mild and warm 545 temperatures and re-expand in response to heat. Notably, unlike in plants, this response in CHO-K1 546 cells did not correlate with significant changes in total nuclear phyB-GFP fluorescence (Fig. 7b), 547 suggesting that heat could increase phyB condensation propensity rather than elevate phyB protein 548 levels. 549 The speckles formed by phyB when co-transfected with either PIF4 or PIF7 were smaller and less 550 amenable to quantitative analysis than those observed with PIF3, but an increase in phyB speckle 551 size in response to heat was still detectable for phyB combinations with either PIF4 or PIF7 (Fig. S6). 552 **High-content automated quantitative analysis of phyB speckle response to temperature** 553 We validated our findings using a fully automated, high-content quantitative approach (Fig. S4). This 554 methodology has two significant advantages for the study of complex patterns because automation 555 minimises the possibility of operator biases and the high-content approach allows for the 556 simultaneous analysis of thousands of CHO-K1 cells. The results confirmed an increase in 557 condensate size at 37°C (Fig. 7c). 558 To investigate whether this temperature-dependent assembly requires thermal reversion, we 559 analysed the constitutively active phyB [Y276H] mutant, which lacks thermal reversion (Su & Lagarias, 560 2007; Hu _et al._, 2009; Wang _et al._, 2024). When co-expressed with PIF3, the size of phyB [Y276H] 561 condensates also increased with temperature indicating that the enlargement observed at 37°C 562 cannot be explained simply by altered thermal reversion dynamics (Fig. 7d). In CHO-K1 cells single 14 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 563 transfected with phyB [Y276H] (no PIF3 co-expression), the speckles also increase in size with 564 temperature (Fig. 7e), suggesting this behaviour is likely an intrinsic property of phyB. 565 We next examined PIF3 speckles that co-localise with either phyB or phyB [Y276H] . These speckles 566 increased in size between 28°C and 37°C, showing the same behaviour as phyB speckles (Fig. 7f). 567 Conversely, PIF3 speckles did not enlarge under heat when phyB was absent (single transfected 568 cells) (Fig. 7g), indicating that PIF3 is not the driver of the response to heat. 569 **Genetically impaired phyB NB formation reduces growth inhibition under heat** 570 The results above demonstrate that NB enlargement and hypocotyl growth inhibition co-occur 571 under heat stress. To move beyond correlation, we investigated whether genetically reducing NB 572 size lowers growth inhibition by comparing hypocotyl elongation in the wild type against single _pch1_, 573 _pchl_ and double _pch1 pchl_ mutants exposed to heat. The _pch1 pchl_ double mutation reduced NB 574 formation in seedlings exposed to high-irradiance R at 37°C (Fig. 8a). In seedlings exposed to a FR 575 pulse to minimise phyB activity followed by darkness at 28°C, hypocotyl growth was unaffected by 576 the mutations (Fig. 8b). However, under high-irradiance R (maximising phyB activity) at 37°C, _pch1_, 577 _pchl_, and _pch1 pchl_ seedlings elongated significantly more than the wild type (Fig. 8b). 578 Establishing direct causality in condensate studies is inherently challenging, as it is "often difficult (if 579 not impossible) to find perturbations that only affect the condensate-forming behaviour while 580 sparing all other functions of the macromolecules involved" (Alberti _et al._, 2025). PCH1 and PCHL 581 are known to exert three primary effects: stabilising the phyB Pfr form by reducing thermal 582 reversion, enhancing phyB NB formation (even in the absence of thermal reversion), and facilitating 583 the degradation of PIF1 protein (Enderle _et al._, 2017; Huang _et al._, 2019; Cheng _et al._, 2020). Notably, 584 these mutations did not exhibit constitutive hypocotyl growth phenotypes in the absence of phyB 585 activation (e.g., through PIF1-mediated effects), as confirmed by our controls exposed to FR at 28°C 586 (Fig. 8b). Furthermore, we minimised the influence of thermal reversion by using high-irradiance 587 continuous R (Burgie _et al._, 2021; Yi _et al._, 2024; Fan _et al._, 2025) ensuring that rapid photochemical 588 reactions outweighed the impact of reversion. Consequently, the increased hypocotyl elongation 589 observed in _pch1_ and _pchl_ mutants under these specific conditions can be directly attributed to 590 impaired NB formation (Fig. 8a-b). ## 591 Discussion 592 While plant growth scales with temperature within permissive ranges, our study demonstrates that 593 rapid hypocotyl growth inhibition under heat stress is not a passive consequence of cellular damage, 594 such as protein denaturation or membrane deterioration. Rather, this inhibition results from a 595 coordinated reversal of the phyB–PIF4–auxin pathway that drives growth during cold-to-warm 596 transitions (Quint _et al._, 2023). This regulatory shift is marked by increased phyB abundance and the 597 formation of distinct heat-induced phyB condensates, a reduction in cotyledonary PIF4 levels, 598 decreased auxin levels, and elevated apoplastic pH, which together reinforce growth inhibition (Figs. 599 1–5). 600 The regulatory impact of heat is remarkably selective. For instance, while PIF4 levels decreased in 601 the cotyledons (the primary site of auxin synthesis) they continued to rise with temperature in the 602 hypocotyl itself (Fig. 4a-d). Despite this local increase, PIF4 activity in the hypocotyl may still be 15 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 603 constrained by phyB-mediated sequestration (Park _et al._, 2018; Pham _et al._, 2018) and a loss of 604 synergism with ARF6 (Oh _et al._, 2014), the levels of which declined under heat (Fig. 2I). In contrast, 605 BES1 (a known component of heat tolerance (Albertos _et al._, 2022)) continued to accumulate and 606 promote growth (Fig. 4I, 3B), highlighting that the heat-induced inhibitory response does not target 607 all growth-promoting pathways equally. Furthermore, levels of the growth-inhibitor HY5 increased 608 under heat, though its maximum physiological effectiveness was observed at lower temperatures 609 (Fig. 4j, 3b). Given that HY5 negatively modulates PIF4 activity(Johansson _et al._, 2014; Gangappa _et_ 610 _al._, 2017), its overall physiological impact likely depends on the stoichiometric interplay between its 611 own abundance and that of its target, PIF4. Similarly, although heat did lower PIF7 protein 612 abundance (Fig. 4g), the reduction in its physiological activity (Fig. 3) likely stems from the depletion 613 of PIF4 precluding the formation of functional PIF4-PIF7 heterodimers(Fiorucci _et al._, 2020) and/or 614 its phosphorylation and sequestration by phyB (Huang _et al._, 2018; Ling _et al._, 2023). 615 The increased abundance and condensation of phyB (Fig. 5) provide a feasible explanation for its 616 enhanced physiological activity under heat (Fig. 3). Condensation may facilitate the activity of a 617 protein by increasing the "dwelling time" with its partners (Ladbury _et al._, 2023). In support of a role 618 of heat-induced condensates, mutations in phyB interactors that impair NB formation result in 619 longer hypocotyls even when combined with a phyB mutation that adopts a constitutively active 620 conformation (Galvão _et al._ ; Enderle _et al._, 2017; Huang _et al._, 2019; Fang _et al._, 2022). More 621 importantly, we observed reduced NB formation and phyB-mediated inhibition of hypocotyl growth 622 in _pch1 pchl_ seedlings under heat (Fig. 8). Overexpression of phyB inhibits hypocotyl growth 623 (Wagner _et al._, 1991), suggesting that the higher phyB protein levels also favour the enhanced phyB 624 mediated growth inhibition under heat. 625 The distinct nature of the heat-induced phyB NBs is highlighted by the fact that it that they formed 626 even following a FR pulse or in a _pch1 pchl_ mutant background (Fig. 6). Because protein 627 concentrations exceeding a critical threshold can trigger the formation of larger condensates via 628 phase separation (Alberti _et al._, 2019), rising phyB levels under heat likely favours this response. 629 However, protein abundance is not the sole driver; heat also appears to enhance the intrinsic 630 propensity of phyB to condense. This was confirmed in mammalian cell, where heat-induced speckle 631 formation occurred even without an increase in phyB protein levels (Fig. 7a–c). In contrast to the 632 decay caused by warm temperatures, the heat-induced increase in phyB NB size does not depend 633 on the occurrence of thermal reversion (Fig. 7d). 634 The inhibitory responses to heat observed here cannot be explained by a simple, linear transmission 635 of a single temperature input through the phyB-PIF4-auxin module. For instance, treatment at 37°C 636 triggered a decay in ARF6 levels (Fig. 2i), an effect not typically considered a consequence of 637 enhanced phyB activity (Pucciariello _et al._, 2018). Furthermore, warm temperatures influence PIF4 638 abundance through pathways independent of phyB (Jung _et al._, 2020), which may also contribute 639 to the observed growth inhibition. These results suggest that the system integrates additional, 640 parallel heat-sensing inputs beyond the canonical phyB-PIF4-auxin regulatory axis. 641 Ecologically, promoting hypocotyl growth at warm temperatures serves as a "foraging" strategy to 642 optimise light capture by outgrowing competing vegetation (Romero-Montepaone _et al._, 2021). 643 However, under extreme heat, this strategy becomes increasingly hazardous. By repressing stem 644 elongation, the plant avoids emerging from the protective shade of the surrounding canopy. This 16 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 645 "umbrella effect", where the seedling remains shielded by the foliage of taller neighbours, helps 646 maintain the plant tissue temperature within a tolerable range and reduce water loss, conferring a 647 vital survival advantage and enhancing resilience under severe thermal stress (Miller _et al._, 2021). ## 648 Acknowledgments 649 650 Supported by _Agencia Nacional de Promoción Científica y Tecnológica_ (ANPCYT, grant PICT-2021-I 651 A-01070 to JJC), University of Buenos Aires (grant 20020170100505BA to JJC) and _Fundación_ 652 _Instituto Leloir_ (institutional grant to JJC). LB received a fellowship from Deutscher Akademischer 653 Austauschdienst (DAAD) to conduct work with mammalian cells in Düsseldorf, Germany, and the 654 Deutsche Forschungsgemeinschaft (DFG, German Research) under the Germany Excellence Strategy 655 (CEPLAS-EXC-2048 project no. 390686111) to M.D.Z. We thank Stefanie Weidtkamp-Peters (Center 656 for Advanced Imaging (CAi) at Heinrich-Heine-University Düsseldorf) for providing access to the 657 Revvity Operetta CLS high-content screening automated microscope system (DFG-INST 208/760-1 658 FUGG), Stefanie Kuschel for mammalian cell culture technical support, and Hannes M. Beyer and 659 Jonas Schön (Institute of Synthetic Biology, Heinrich-Heine-University Düsseldorf) for helpful 660 discussions. 661 During the preparation of this work, the authors used Google Gemini to improve the choice of 662 words. The authors reviewed and edited the output as needed and take full responsibility for the 663 content of the published article. 664 ### 665 Author contributions 666 667 Investigation, L.B., C.S.K., M.B.D.-L., G.M. and M.E.P.; conceptualization, J.J.C.; writing, J.J.C. with 668 input from L.B., C.S.K., M.B.D.-L., G.M., M.E.P., A.B.H, and M.D.Z. ; funding acquisition, M.D.Z., and 669 J.J.C., visualization, L.B., C.S.K. and J.J.C.; formal analysis, L.B., C.S.K., A.B.H. and J.J.C.; supervision, 670 M.D.Z., and J.J.C. 671 ### 672 Declaration of interests 673 674 The authors declare no competing interests. 675 ## 676 References 677 678 **Alberti S, Arosio P, Best RB, Boeynaems S, Cai D, Collepardo-Guevara R, Dignon GL, Dimova R,** 679 **Elbaum-Garfinkle S, Fawzi NL,** _**et al.**_ **2025** . Current practices in the study of biomolecular 680 condensates: a community comment. _Nature Communications_ **16** : 7730. 681 **Alberti S, Gladfelter A, Mittag T** . **2019** . 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Wandering between hot and cold: temperature dose969 dependent responses. _Trends in Plant Science_ **27** : 1124–1133. 970 971 23 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 972 973 **Figure 1.** **Impaired growth inhibition by hot temperatures in auxin-related mutants** . 974 (a) Representative of Col-0 seedlings grown for 9 h at the indicated temperatures. The base of the 975 pictures corresponds to the top of the hypocotyl at the beginning of the treatments. 976 (b) Compared to Col-0 seedlings, the auxin-related _sav3_, _yucQ_, _saur19–24_ and _saur61–68, 75_ 977 mutants show impaired promotion of hypocotyl growth by warm temperature and lack heat 978 induced growth inhibition. 979 (c) Exogenous application of the synthetic auxin Picloram (Table S1) promotes hypocotyl growth but 980 does not fully restore heat-inhibited growth. 981 The seedlings were grown at 22°C, transferred to the indicated temperatures 1 h after the beginning 982 of the photoperiod of the fourth day, and photographed 0 and 9 h after the beginning of 983 temperature treatments to measure hypocotyl growth. Each datum point is the mean ±SE of 6 984 biological replicates pooled from three independent experiments. Asterisks denote statistical 985 significance of linear or quadratic regression lines and of the differences between plants treated 986 with Picloram and controls at a given temperature: ***, P <0.001; **, P <0.01; *, P <0.05. 987 988 24 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 989 990 **Figure 2. Hot temperatures reduce auxin abundance, perception and signalling** 991 (a-b) Auxin quantification with the FRET sensor AuxSen in hypocotyl cells (a) or by a colorimetric 992 assay in whole seedlings (b) show a peak at warm temperature and a decline at hot temperatures. 993 (c) Confocal analysis of DII-VENUS in hypocotyl cells shows a minimum at warm temperature 994 (indicating high auxin signalling) and an increase at hot temperatures (indicating low auxin 995 signalling). 996 (d-f) Confocal analysis of _DR5:GFP_ reporters in hypocotyl cells (d-e, two different reporters) or 997 bioluminescence assays with the _DR5:GUS_ reporter (f) indicate maximal ARF transcriptional activity 998 at warm temperatures and reduced activity at hot temperatures. 999 (g) Meta-analysis of the expression of genes corresponding to the GO term “response to auxin” 1000 shows a peak at warm temperatures and a decline at hot temperatures. 25 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1001 (h) Confocal analysis of TIR1-mCitrine in hypocotyl nuclei indicates increased abundance of the TIR1 1002 auxin receptor at warm temperatures and stable values at hot temperatures. 1003 (i) Confocal analysis of ARF6-GFP in hypocotyl nuclei indicates increased abundance of ARF6 1004 transcription factor at warm temperatures and a decrease at hot temperatures. 1005 (j) Confocal analysis of the apoplastic pH reporter Apo-pHluorin shows increased pH at hot 1006 temperatures. 1007 The seedlings were grown at 22°C, transferred to the indicated temperatures 1 h after the beginning 1008 of the photoperiod of the fourth day, and harvested 4 h after the beginning of temperature 1009 treatments. Scale bars: 5 µm (a) or 50 µm (c-e, g, j, i). Each datum point is the mean ±SE of 12-16 1010 seedlings (a, c-f, h-j) or 3 biological replicates (b), pooled from three independent experiments. Data 1011 of meta-analysis (I) are the mean ±SE of log2 Fold Change of expression for 48 genes responsive to 1012 auxin (GO:0009733) that responded significantly to temperature. Asterisks denote statistical 1013 significance of linear or quadratic regression lines and between control: ***, _P_ <0.001; **, _P_ <0.01; 1014 *, _P_ <0.05. 1015 1016 26 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1017 1018 **Figure 3. Functional analysis of key thermomorphogenic genes indicates a reversal of their** 1019 **physiological impact at supra-optimal temperatures.** 1020 (a) Hypocotyl growth of Col-0 wild-type seedlings and _phyB_, _pif7_, _pif4_, _pif5_, _pif3_, _bes1_ and _hy5_ 1021 mutants exposed to different temperatures. 1022 (b) Impact of the _PHYB, PIF7, PIF4, PIF5, PIF3, BES1_ and _HY5_ genes on hypocotyl growth calculated 1023 by using a two-equation mathematical model incorporating the wild-type and null mutant data 1024 shown in A, as described in the Methods section. 1025 The seedlings were grown at 22°C, transferred to the indicated temperatures 1 h after the beginning 1026 of the photoperiod of the fourth day, and photographed 0 and 9 h after the beginning of 1027 temperature treatments. Each datum point is the mean ±SE of 8-14 biological replicates pooled from 1028 three independent experiments. Asterisks denote statistical significance of linear or quadratic 1029 regression lines: ***, _P_ <0.001; **, _P_ <0.01; *, _P_ <0.05. 1030 1031 1032 27 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1033 1034 **Figure 4. Reduced PIF4 protein abundance at hot temperatures** 1035 (a-d) Confocal analysis of the fluorescence driven by the _pPIF4:PIF4-GFP_ reporter in the cotyledons 1036 (a, c) or the hypocotyl (b, d) in seedlings exposed to different temperatures for 4 h (a, b) or 8 h (c, 1037 d). 1038 (e) Protein blot of PIF4-HA in seedlings exposed to different temperatures for 4 h. 28 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1039 (f) Luminescence driven by the _pPIF4:PIF4-LUC_ reporter in seedlings exposed to different 1040 temperatures for 4 h. 1041 (g) Protein blot of PIF7-HA in seedlings expressing the pPIF7:PIF7-HA transgene and exposed to 1042 different temperatures for 4 h or 8 h. 1043 (h) Levels of the _PIF7_ transcripts measured by qRT-PCR in seedlings exposed to different 1044 temperatures for 4 h. 1045 (i-j) Confocal analysis of the fluorescence driven by the _p35S:BES1-GFP_ reporter (i) or the _pHY5:HY5-_ 1046 _YFP_ reporter (j) in seedlings exposed to different temperatures for 4 h or 8 h. 1047 The seedlings were grown at 22°C, transferred to the indicated temperatures 1 h after the beginning 1048 of the photoperiod of the fourth day, and harvested 4 h or 8 h after the beginning of temperature 1049 treatments. Scale bars: 50 µm (a-d, i-j). Each datum point is the mean ±SE of 14 seedlings (a-d, i-j) 1050 or 3 (e, i), 6 (f), or 4 (g) biological replicates pooled from three (a-d, i-j) or two (e-i) independent 1051 experiments. Asterisks denote statistical significance of linear or quadratic regression lines: ***, _P_ 1052 <0.001; **, _P_ <0.01; *, _P_ <0.05. 1053 1054 29 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1055 1056 **Figure 5. Increased phyB NB size at hot temperatures** 1057 (a-c) Confocal analysis of the area of the NBs in hypocotyl cells driven by _pUBQ10:PHYB-YFP_ (a), 1058 _p35S:PHYB-GFP_ (B) or _pPHYB:PHYB-YFP_ (c). 1059 (d-f) Confocal analysis of the nuclear fluorescence in hypocotyl cells driven by _pUBQ10:PHYB-YFP_ 1060 (D), _p35S:PHYB-GFP_ (E) or _pPHYB:PHYB-YFP_ (f). 1061 (g-h) Protein blot of PHYB-GFP in seedlings expressing the _p35S:PHYB-GFP_ (g) or _pPHYB:PHYB-YFP_ 1062 (h) transgenes. 1063 The seedlings were grown at 22°C, transferred to the indicated temperatures 1 h after the beginning 1064 of the photoperiod of the fourth day, and harvested 4 h after the beginning of temperature 1065 treatments. Scale bars: 5 µm (a-c) or 20 µm (d-f). Each datum point is the mean ±SE of 12-16 1066 seedlings (a-f) or 4 biological replicates (g-h) pooled from three (a-f) or two (g-h) independent 1067 experiments. Asterisks denote statistical significance of linear or quadratic regression lines: ***, _P_ 1068 <0.001; **, _P_ <0.01; *, _P_ <0.05. 1069 1070 30 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1071 1072 **Figure 6. High temperatures promote phyB NB formation despite FR-induced inactivation** 1073 (a-d) Confocal analysis of the area of the NBs driven by _p35S:PHYB-GFP_ in seedlings exposed to the 1074 indicated light and temperature conditions. In D (left) the transgene is expressed in the _pch1 pchl_ 1075 background. 1076 (e) Impact of the _PHYB_ gene on hypocotyl growth calculated by using a two-equation mathematical 1077 model incorporating the wild-type and _phyB-9_ null mutant data, as described in the Methods 1078 section. 1079 The seedlings were grown at 22°C and, 1 h after the beginning of the fourth day, were transferred 1080 to a pulse of R (5 min, 35 μmol m [-2] s [-1] ) followed by darkness, cycles of R (5 min, 35 μmol m [-2] s [-1] ) and 1081 darkness (55 min) repeated every hour (the first one immediately after the end of 1 h white light), 1082 or a pulse of FR (5 min, 15 μmol m [-2] s [-1] ) followed by darkness. These light / dark conditions were 1083 combined with the indicated temperatures. Scale bars: 5 µm (a-d). Data are the mean ±SE and 1084 individual values of 8-16 seedlings (a-d) or 8 biological replicates (e), in each case poled from three 1085 independent experiments. Asterisks denote statistical significance of ANOVA followed by Tukey 1086 tests (a-c) or Student’s t tests (d-e): ***, _P_ <0.0001; **, _P_ <0.01; *, _P_ <0.05. 1087 1088 31 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1089 1090 **Figure 7. High temperatures enhance phyB condensate size in mammalian cells.** 1091 (a-b) Confocal analysis of the area of the NBs (a) and total nuclear fluorescence (b) driven by 1092 _pSV40:PHYB-eGFP_ in CHO-K1 cells co-transfected with _pSV40:PIF3-eCerulean._ 1093 (c) Fully automated, high-content quantitative confocal analysis of the area of the speckles driven 1094 by _pSV40:PHYB-mCherry_ co-transfected with _pSV40:PIF3-eGFP._ Both, nuclear and cytoplasmatic 1095 speckles are included in the analysis. 32 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1096 (d-e) Fully automated, high-content quantitative confocal analysis of the area of the speckles of 1097 _pSV40:PHYB_ _[Y276H]_ _-mCherry_ in CHO-K1 cells co-transfected with _pSV40:PIF3-eGFP_ (d) or single 1098 transfected with _pSV40:PHYB_ _[Y276H]_ _-mCherry_ (e) _._ 1099 (f) Correlation between the size of the speckles driven by _pSV40:PIF3-eGFP_ and the speckles driven 1100 by either _pSV40:PHYB-mCherry_ or _pSV40:PHYB_ _[Y276H]_ _-mCherry_ co-transfected in CHO-K1 cells. 1101 (g) The size of the speckles driven by _pSV40:PIF3-eGFP_ do not increase in response to heat in single 1102 transfected cells. 1103 CHO-K1 cells were exposed to 15-min R followed by 60 min of darkness at the indicated 1104 temperatures. Scale bars: 20 µm (a). Each datum point is the mean ±SE of 8–10 biological replicates 1105 (c-e), the mean ±SE and individual values of 12-20 biological replicates (a-b, g), or individual values 1106 (f) pooled from two (a-c, g) or three (d-e) independent experiments. Please refer to Figure S4 for an 1107 example showing the number of cells analysed in an individual experiment contributing to c-e. 1108 Asterisks denote statistical significance of ANOVA followed by Tukey tests (a-b) or of linear or 1109 quadratic regression lines (c-g): ***, _P_ <0.0001; **, _P_ <0.01; *, _P_ <0.05. 1110 1111 33 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764838; this version posted November 9, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.06.764838) (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 a CC-BY-NC-ND 4.0 International license.](http://creativecommons.org/licenses/by-nc-nd/4.0/) 1112 1113 **Figure 8.** **Genetically impaired phyB NB formation reduces growth inhibition under heat.** 1114 (a) Confocal analysis of the area of the NBs driven by _p35S:PHYB-GFP_ in seedlings of the wild-type 1115 and _pch1 pchl_ mutant backgrounds exposed to high-irradiance continuous R (175 μmol m [-2] s [-1] ) at 1116 37°C. 1117 (b) Hypocotyl growth in seedlings of the wild-type and _pch1 pchl_ mutant exposed to FR followed by 1118 darkness at 28°C (as a control showing no effects of the mutations under conditions of minimal phyB 1119 activity) or high-irradiance continuous R at 37°C. Note different scales. 1120 The seedlings were grown at 22°C and, 1 h after the beginning of the fourth day, were transferred 1121 to a pulse of FR (5 min, 15 μmol m [-2] s [-1] ) followed by darkness at 28°C or high-irradiance continuous 1122 R (175 μmol m [-2] s [-1] ) at at 37°C. Scale bars: 5 µm (a). Data are the mean ±SE and individual values of 1123 4-12 biological replicates (a) or 11-12 seedlings (b), in each case pooled from two independent 1124 experiments. Asterisks denote statistical significance of ANOVA followed by Tukey tests (a) or 1125 Student’s t tests (b): ***, _P_ <0.0001. 34