AUXIN RESPONSE FACTOR thermostability

Domain: biology
Published: 2026-03-27
Editorial outcome: major_revision

Abstract
Plants use the plant hormone auxin to incorporate environmental cues into their growth and development to shape the final form. Temperature is an important modulator of all aspects of plant function and growth. In this work, we uncover temperature-regulated accumulation and solubility of members of the AUXIN RESPONSE FACTOR transcription factor family. We determine that ARF7 and ARF19 proteins rapidly hyperaccumulate in response to elevated temperature. Furthermore, we find that diffuse concentrations of ARF protein increase under elevated temperature, consistent with increased solubility. Temperature-driven ARF hyperaccumulation is not fully dependent on the well-established temperature response pathways. We find that natural variation in thermoregulated ARF accumulation is correlated with thermomorphogenesis, suggesting that this is a dial switch in plant temperature response. Regulated ARF thermoaccumulation provides a layer of complexity in shaping and plant growth and form, allowing plants to respond rapidly and persistently to elevated temperatures by modulating levels of nuclear ARF protein accumulation.

Introduction
Although plants start their lives with a simple body plan, to adjust their shape to the prevailing conditions, they must incorporate environmental information into developmental and physiological decision-making. A critical cue in this process is environmental temperature, which provides critical information about daily and seasonal rhythms and the risk of stressful conditions. Plants respond to elevated temperature in a suite of growth changes characterized as thermomorphogenesis1. In addition, a myriad of physiological changes enables plants to adjust to changing temperatures2,3. Understanding the mechanisms used by plants to optimize their body shape and physiological responses in response to increased temperature is particularly important as our global temperatures continue to rise.

Elevated temperatures input through multiple pathways, seemingly converging on increased auxin biosynthesis4,5. Indeed, functional auxin receptors TIR1/AFB, which work in conjunction with IAA3, and IAA19 co-receptors, are required for thermomorphogesis6–9. Auxin-driven association of these Aux/IAA repressors with TIR1/AFB F-box proteins promotes their polyubiquitylation and degradation, relieving ARF proteins from their repression10. Auxin transcriptional output is driven by the AUXIN RESPONSE FACTOR (ARF) transcription factor family, which is repressed by the Aux/IAA proteins in the absence of auxin. Although these auxin-signaling components are necessary for thermomorphogenesis, whether ARFs act simply as interpreters of elevated auxin levels or if they also represent direct targets of temperature cues remains poorly understood.

In addition to Aux/IAA-mediated repression, activity of ARF7 and ARF19, and perhaps additional ARFs, is also attenuated by protein condensation11, which is a rapid and reversible compartmentalization of proteins12. Protein condensation is a concentration-dependent process in which proteins in excess of a saturation concentration are stored in dense bodies, or condensates12. This saturation concentration is modified by environmental conditions, post translational modifications, and interaction partners, allowing for rapid and dynamic compartmentalization of cellular components. ARF7 and ARF19 condensation is regulated by unknown developmental regulators11, interaction with MCTP proteins13, and condensate movement14. Further, regulated ARF accumulation is increasingly recognized as a regulator of auxin response15–17.

Here, we uncover temperature-regulated changes in ARF protein accumulation, stability and solubility. Further we find that ARF7 and ARF19 temperature-regulated accumulation correlates with thermomorphogenesis in natural Arabidopsis accessions. This ability of ARF proteins to intrinsically respond to rapid changes in temperatures adds a new level of complexity to the auxin transcriptional response and incorporation of temperature cues into plant growth.

Results
Class-A ARF protein accumulation is dynamically regulated by temperature
ARF protein stability is emerging as an important regulator of auxin response15,18. To understand regulators of ARF protein stability, we developed a custom antibody, raised against the ARF7 PB1 domain (Figure S1), which recognizes both ARF7 and ARF19 proteins. Using this antibody, we found that endogenous levels of ARF7 and ARF19 rise with increasing temperatures (Fig. 1a). ARF7 and ARF19 transcripts are unaffected by temperature changes (Supplementary Table 1) and are thus unlikely to be the driver of differential protein accumulation at varying temperatures. Further, when driven behind constitutive promoters, ARF7 and ARF19 protein accumulation varies with temperature (Fig. 1b–f, Figure S2a), suggesting that translation and/or stability of these proteins is temperature-regulated.

In Arabidopsis, there are five Class-A ARFs (Fig. 1f), which are thought to positively drive auxin-responsive gene expression10. ARF7 and 19 are closely-related, with ARF5 as the next most-closely related to ARF7 and 19. ARF6 and 8 are additional Class-A ARFs in a separate clade from ARF7, 19, and 519. We examined these additional Class-A ARFs and found that ARF5, ARF6, and ARF8, similar to ARF7 and ARF19, undergo a dramatic increase in accumulation in response to elevated temperature (Fig. 1g). Thus, all Arabidopsis Class-A ARFs display thermoresponsive protein accumulation. Because our lab has many ARF7- and ARF19-related resources11,14,15,20–22, including our newly-developed custom antibody (Figure S1), we proceeded with studies focused primarily on ARF7/19.

ARF protein stability is regulated by temperature
To assess ARF protein stability, we developed a flow cytometry-based ratiometric assay. In this assay in which protoplasts transfected with a ratiometric reporter encoding a P2A self-cleaving peptide (pUBQ10:mNEON-ARF19-P2A-mScarlet) allowed the measurement of two separate fluorescent proteins encoded by a single mRNA transcript (Fig. 2a, b). Using this assay, we found that ARF19 displayed increased stability at 32°C compared to 22°C (Fig. 2b).

We found that ARF7/19 protein displays a longer half-life at 32°C compared to 22°C using a cycloheximide chase experiment (Fig. 2c, d). We then treated seedlings at elevated temperature for four hours prior to a four-hour recovery at 22°C, with samples taken before and after elevated temperature exposure and after the recovery period (Figure S2b–e). In these assays, ARF7 and ARF19 protein accumulation largely returned to baseline levels after transition back to 22°C. This incomplete recovery suggests that protein levels do not revert to their original levels at the same rate that they accumulate. Overall, our data show that ARF7 and ARF19 protein accumulation is dynamically regulated as a function of temperature; these effects are driven by altered protein stability.

Next, we examined protein degradation mechanisms that might control temperature-regulated ARF protein accumulation. To date, we know that ARF protein accumulation is regulated by the 26S proteasome18 and by autophagy23. ARF7 levels increased at elevated temperature in both the absence and presence of a proteasome inhibitor (Fig. 2e), suggesting that 26S proteasomal activity is not a major driver of the effects of temperature on ARF stability. Aligned with this result, we found that ARF7/19 accumulation was equally temperature-regulated in wild type and in the aff1 mutant (Figs. 2f, S3b), which is defective in an E3 ubiquitin ligase that targets ARF7 and ARF19 to the proteasome24. Further, temperature effects on ARF7 accumulation were unaffected by the autophagy inhibitor Concanamycin A (Figure S3a), suggesting that this pathway is also unnecessary for the altered ARF accumulation in response to changing temperatures. Finally, to determine if the chaperone protein HSP90 impacted ARF temperature-regulated stability, we assessed ARF7 protein levels in the presence of HSP90 inhibitor Glendamycin A (Figure S3c), finding that ARF7 accumulation remained thermoresponsive in the presence of this inhibitor, suggesting that HSP90 is dispensable for temperature effects on ARF accumulation. Given these results, we postulate that ARF thermostability could be regulated by alternative mechanisms such as interaction with chaperones other than HSP90 or allosteric regulation not captured in the experiments performed.

ARF19 thermoresponsive accumulation is conferred by the DBD and MR regions
We were curious what aspects of ARF7 and ARF19 drove thermoresponsive protein accumulation. To determine which ARF19 regions confer thermoaccumulation properties, we examined ARF19 truncations consisting of the DNA binding Domain (DBD, ARF191–354), the PB1 domain (ARF19958–1086), and the PB1 domain plus the middle region (MR) of the protein (MR+PB1, ARF19355–1086) (Fig. 3a). We found that the ARF19 PB1 domain does not display thermoresponsive accumulation (Fig. 3b). In contrast, both the ARF19 DBD and the ARF19 MR+PB1 displayed thermoresponsive accumulation (Fig. 3b). Because these two proteins share no residues in common, we conclude that more than one region of the protein bolsters protein stability at elevated temperatures.

We next sought to identify other residues that could confer stability using an in vivo mutagenesis screen. We randomly mutagenized the pUBQ10:YFP-ARF19 plasmid using an error prone polymerase25 followed by bulk Arabidopsis transformation with these variants (Fig. 3c). We then screened transformants on coverslip-bottom culture plates and a heated stage, identifying ARF19W330L and ARF19R297W variants that failed to display thermoresponsive accumulation (Figure S4). Both mutations are in conserved residues within the ancillary domain of the DBD (Fig. 3d). These mutations are near a region recently reported to regulate ARF stability16. To confirm that these variants impact ARF stability, we assessed variant thermostability using the ratiometric reporter in protoplasts (Fig. 2a), finding that each variant displays attenuated thermoresponsive stability (Fig. 3f).

In the ARF19 protein model, W330 was buried within the ancillary domain of the DBD and had a three-way polar interaction with R331 and E51 (Fig. 3e). ARF19W330L is likely to break the polar interaction with E51. R297 occurs within a beta-sheet of a five-stranded beta-barrel of the ancillary domain and forms a polar contact with E353 at the end of a predicted alpha-helix (Fig. 3e). In ARF19R297W, the polar contacts from the R-group are likely to be ablated. Further, the substitution of a positively charged arginine residue with a bulky aromatic tryptophan could disrupt intramolecular interactions in this region of the protein. Altogether, because thermoresponsive accumulation is displayed by more than one ARF protein region, we speculate that multiple factors contribute to ARF thermostability.

To assess the functional role of ARF19 temperature-responsive accumulation, we created stable rescue lines by transformation arf7 arf19 with UBQ10:YFP-ARF19, UBQ10:YFP-ARF19W330L, or UBQ10:YFP-ARF19R297W. We found that, similar to what we found in our screen (Figure S8), the ARF19W330L and ARF19R297W variants displayed less thermoresponsive protein accumulation than wild type ARF19 (Fig. 3g). However, these stable lines with these variants also displayed decreased accumulation at 22°C when compared to wild type protein accumulation, suggesting that these variants affect ARF19 protein stability at both ambient and elevated temperature. Expressing UBQ10:YFP-ARF19 in arf7 arf19 mostly rescued its thermomorphogenesis phenotype (Fig. 3h); this partial rescue is likely due to the fact that this was in the double mutant background, and we rescued with only ARF19. Wild type ARF19, ARF19W330L and ARF19R297W each similarly mostly rescued the long hypocotyl developmental phenotype of arf7 arf19 at 22°C, suggesting that at this temperature, both variants accumulate sufficient protein to compensate for loss of genomic ARF19 (Fig. 3h). In contrast, neither UBQ10:YFP-ARF19W330L nor UBQ10:YFP-ARF19R297W rescued arf7 arf19 thermomorphogenic growth defects (Fig. 3i), suggesting that temperature-regulated ARF19 accumulation contributes to temperature-responsive growth. After finding that ARF protein thermoresponsive stability activity is encoded in both the DBD and disordered middle region, we sought to understand how ARF proteins fit into known temperature pathways, and whether temperature impacted its nucleo-cytoplasmic partitioning.

Temperature-responsive ARF condensation
Given the established relationship between ARF7 and ARF19 function and condensation11, we examined the effects of varying temperatures on ARF7 and 19 condensation and nuclear localization. In phase-separating systems, temperature frequently regulates solubility12 (Fig. 4a). Because diffuse/soluble ARF7 and 19 are localized to the nucleus whereas ARF condensates are localized to the cytoplasm11,26, we examined whether increasing temperature would result in increased soluble/nuclear ARF protein. This question is particularly important because in phase separating systems, increased overall protein concentrations do not necessarily result in increased ‘active’ protein when the protein in the dilute phase is the active form of protein. In these systems, the concentration of dilute phase protein is buffered against changes in overall protein levels. We found that the concentration of nuclear (i.e., diffuse) ARF protein increases with increasing temperatures (Fig. 4b–d). In time course imaging of hypocotyls exposed to elevated temperature, we observe a rapid increase in nuclear signal (8 min post-exposure) prior to the occurrence of cytoplasmic condensates (~31 min post-exposure), consistent with a model in which elevated temperature conditions allow for increases in soluble/nuclear ARF protein (Fig. 4e, f). Congruent with this result, Borniego et al. (see companion article) demonstrate that ARF7 and ARF19 display increased solubility at elevated temperature when expressed in yeast.

In a phase-separating system12, environmental conditions such as temperature frequently affect the saturation concentration in the system (Fig. 4a). In these systems, the concentration of the dilute phase equals that of the saturation concentration of the system, such that the dilute phase concentration can change with environmental conditions. For the ARF proteins, the dilute ARF protein resides in the nucleus where it is transcriptionally active11,14,26. Thus, the concentration of nuclear ARF protein likely correlates with the solubility of the protein.

To test whether increased ARF solubility and nuclear concentration affected transcriptional output, we directly examined ARF7 transcriptional output using a protoplast-based reporter of transcriptional activity, in which the ARF7 DBD has been replaced with the GAL4 DBD in a FrankenARF protein fused to mVenus22 (Fig. 4g). In the same plasmid, the GAL4 UAS is upstream of an mScarlet reporter to allow for ratiometric analysis of ARF7 transcriptional output. Examination of ARF7 activity in protoplasts at 22°C and 32°C reveals increased transcriptional output at this temperature, consistent with elevated ARF7 levels in the nucleus at this temperature (Fig. 4h).

Our data is consistent with the possibility that ARF7 and 19 display upper critical solution temperature (UCST) behavior (Fig. 4a), often displayed by Prion-like domain (PrLD)-containing proteins27,28. In UCST phase separation systems, molecules such as ARF7 and 19 display increased solubility at elevated temperature and thus the dilute concentration of the molecule increases with increasing temperature (Fig. 4a). Enthalpy-driven UCST behavior can be mediated by aromatic side chain associations. Analysis of the ARF7 and ARF19 intrinsically disordered regions reveals residues compatible with UCST behavior (Figures S5, S6). Indeed, Borniego et al. (accompanying manuscript) observe temperature-regulated ARF7 and ARF19 solubility in root tissues and in yeast. In sum, our data are consistent with a model in which temperature regulates the levels of nuclear ARF7 and ARF19 protein not only by altering protein stability but also by increasing protein solubility.

ARF7 and ARF19 thermoresponsive accumulation is not fully dependent on known thermosensing pathways
PHYTOCHROME INTERACTING FACTOR 4 (PIF4) is a central signaling hub of thermomorphogenesis and drives the thermoresponsive expression of auxin biosynthesis genes YUCCA8 (YUC8) and YUC9. ELONGATED HYPOCOTYL 5 (HY5) acts antagonistically to PIF4 by competing for YUC8 binding sites29–32. The thermosensor EARLY FLOWERING 3 (ELF3) also negatively regulates PIF433–36. The light receptor phyB is a thermosensor that interacts with PIF436,37. Together, these pathways integrate temperature cues to plant physiological adaptations to temperature5. Ultimately, these pathways result in elevated auxin biosynthesis through the YUCCA family (Fig. 5a). To determine whether temperature-regulated auxin biosynthesis was required for our observed ARF7 thermoregulated protein accumulation, we examined ARF7 protein levels in the yucQ and 35S:YUC backgrounds. We found that ARF7 temperature-regulated accumulation was similar to wild type (Figure S7a, b).

To ascertain if ARF thermostability was regulated by PIF4 and HY5, we measured ARF7 protein levels in the hy5 and phyB pifQ mutants, finding that ARF7 thermoresponsive accumulation was not different from wild type in these backgrounds (Fig. 5b, c). We further assayed ARF7 protein levels in the elf3 elf4 mutant background and in a line overexpressing ELF3 (35S:ELF3). ARF7 thermoresponsive accumulation was unaffected in the elf3 elf4 mutant (Fig. 5d). Conversely, ELF3 overexpression resulted in slightly elevated ARF at all temperatures (Fig. 5d), suggesting a more complicated relationship between ARF7 stability and ELF3, which plays roles not only in temperature sensing36, but also in circadian clock regulation38. To further investigate the effects of PhyB and PIFs on ARF thermoresponsive accumulation, we assessed temperature-regulated ARF7 accumulation in phyB and pifQ, finding that in these mutants, ARF7 thermoresponsive protein accumulation is similar to wild type in these mutant backgrounds (Fig. 5e, f).

Elevated temperature results in elevated auxin levels through the activities of known temperature sensing pathways5. To separate the effects of increased auxin from ARF7/ARF19 activity, we examined temperature-responsive hypocotyl elongation of wild type, the auxin overproducing line 35S:YUC139, and arf7 arf19 with and without the auxin inhibitors L-Kynurenine40 and Yucasin41. Without inhibitors, both auxin overproduction and loss of ARF7 and 19 activity dampened thermomorphogenic response at 28°C and 32°C (Figure S8b, c). In 35S:YUC1, auxin levels were likely saturated, limiting further temperature-inducted elongation. Inclusion of auxin biosynthesis inhibitors did not fully block thermomorphogenic hypocotyl elongation in either wild type or in the 35S:YUC1 line (Figure S8b, c). Conversely, inclusion of auxin biosynthesis inhibitors diminished arf7 arf19 hypocotyl thermomorphogenesis (Figure S8b, c). The auxin reporter, DII-Venus confirmed that the auxin inhibitors blocked auxin biosynthesis (Figure S8d, e). The dynamic nature of temperature-regulated Class-A ARF accumulation (Fig. 1), condensation (Fig. 4) and roles in thermomorphogensis (see Borniego et al., companion article) suggests roles for ARF proteins in temperature sensing that are not directly reliant on their roles interpreting auxin levels.

Natural variation in temperature-regulated ARF stability associated with changes in thermomorphogenesis
We were curious if ARF stability was important for thermomorphogenesis in other accessions. Therefore, we leveraged naturally-occurring genetic variation within Arabidopsis thaliana to see if different levels of ARF stability had consequences on hypocotyl thermomorphogenesis. We chose 15 accessions that have evolved in diverse environments (Fig. 6a) and compared hypocotyl elongation at 22°C and 28°C. We found a spectrum of hypocotyl elongation in response to the increased temperature (Fig. 6b). We then assayed thermoresponsive ARF7/19 protein accumulation within these accessions using immunoblotting. Some accessions, such as Ler-1, do not display a sharp accumulation in ARF7/19 at elevated temperature whereas other accessions, such as MNF-Che-47, display a robust increase in ARF7/19 accumulation at elevated temperature (Fig. 6c). We then analyzed the relationship between thermomophogenic hypocotyl elongation and thermoresponsive ARF protein accumulation using linear regression and found a correlation between thermoresponsive ARF7/19 protein accumulation and thermormophogenic hypocotyl elongation (Fig. 6d). The strength of this correlation was unexpected, considering the multiple pathways regulating thermomorphogenesis and the broad differences amongst accessions; however, this data suggests that temperature-regulated stability of the ARF7/19 transcription factors, which mediate auxin transcriptional output, contributes to the thermomorphogenic response across Arabidopsis accessions.

Methods
Plant Growth Conditions
Thermomorphogenesis assays: Seedlings were stratified 4°C overnight and plated on Plant Nutrient Media43 solidified with 0.6% agar and supplemented with 0.5% (w/v) sucrose. To examine the effects of auxin inhibitors on hypocotyl elongation, seedlings were grown for one day under yellow-filtered light prior to grown under dark conditions at 22°C to encourage hypocotyl elongation. Seedlings were then transferred to media containing a mock treatment (DMSO), or 50µM Yucasin (gift from Jerry Cohen) and 10 µM L-Kynurenine (Millipore Sigma). Yucasin and L- Kynurenine (L-kyn) are auxin biosynthesis inhibitors that respectively block the enzymatic activities of YUCCA and TAA40,41. Seedlings on mock or auxin inhibitor treatment were incubated at 22°C, 28°C, or 32°C vertically under continuous yellow-filtered light for an additional 3d prior to imaging and measurement of hypocotyl lengths using FIJI.

For protoplast assays, 7-day-old seedlings were transferred to pots containing Metro-Mix 830 media (Sungro) and placed at 22°C at 16h light for an additional 7–10 days. For the growth of T1 agrobacterium transformants, seeds were placed directly in Metro-Mix 830 at 16h light for 7 days. After 7 days, positive transformants were selected by spraying the seedlings with BASTA. After selection, plants were grown at 16h light until mature. Transformants were harvested as individuals and their progeny were screened.

Vector Construction
pUBQ10:mNEON-ARF19c-P2A-mScarlet3 was generated using the pUBQ-mNEON-P2A-mScarlet3 backbone. pUBQ10:mNeon-P2A-mScarlet (using 5′-AACGCATGCGGAAGCGGAGC-3′ and 5′-AACACTCCCACTTC CAGAGCC-3′) and ARF19 CDS (using 5′-GGAAGTGGGAGTGTTATGAA AGCTCCATCAAATGG-3′ and 5′-GCTTCCGCATGCGTTTCTGTTGAAA GAAGCTGCAGCAG-3′) were linearized via PCR (Invitrogen, SuperFi II) and the ARF19 CDS was integrated into pUBQ10:mNeon-P2A-mScarlet backbone using NEBuilder HiFi DNA Assembly (New England Bioscience). ARF19 variant fragments were generated by twist Bioscience in the pENTR backbone and inserted into the pUBQ10: mNeon-P2A-mScarlet3 backbone using NEBuilder HiFi DNA assembly.

FrARF7 and GAL4 DBD plasmids were generated as previously described22: A gene fragment which encoded the FrARF7 protein, a NOS terminator, and 1000bp of non-coding DNA including a multiple cloning site was synthesized by Twist in the pENTR Gateway compatible backbone. An additional gene fragment encoding 500bp of non-coding DNA followed by 5X Gal4 UAS sites, a minimal CaMV 35S promoter, and mScarlett-I fused to Histone 2B was synthesized and put into the pENTR backbone by Twist. The pENTR FrARF7 backbone was linearized using SacI, and the reporter was PCR amplified to include 20bp overlap with the pENTR FrARF7 multiple cloning site at the 5´ and 3´ ends. The reporter insert was cloned into the linearized FrARF7 vector using NEBuilder HiFi cloning to generate pENTR FrARF7+Reporter clones. The FrARF7+Reporter Entry clone was then cloned into pLCS107, which provided an in-frame mNEON fused to the N-terminus of FrARF7 driven under the UBQ10 promoter and a nos terminator for the mScarlet-I H2B reporter, by gateway cloning. The Gal4 DBD variant was generated by in frame deletions of the ARF7 CDS by PCR linearization and self-assembly with NEBuilder HiFi cloning.

Immunoblot Analysis
Custom polyclonal antibody was generated by injecting rabbits with purified ARF7 PB1 domain21 using a standard 70-day immunization protocol by the company Biomatik.

Fresh tissue from 30 4-day-old seedlings was homogenized using a 2010 Geno/Grinder (SPEX Sample Prep) at 1500 RPM for 30 seconds and then placed at 70°C in 2x NuPage LDS buffer (141mM Tris, 2% Lithium Dodecyl Sulfide, 0.51mM EDTA, 10% glycerol, 0.175mM Phenol Red, 0.22mM Coomassie Blue) and loaded onto a Bolt 8% Bis-tris protein gel (Thermo Scientific). The gel was run at 80V for 15min and then 160 volts for 1h until proper separation of bands was obtained. The gel was transferred to a nitrocellulose membrane (Amersham Protan 0.45µm NC) using a wet-transfer system. Loading control was determined with Ponceau solution (0.1% Ponceau w/v, 5% acetic acid) for 2min, washed in TBS-T solution (200mM Tris, 1.5M NaCl, 0.1% Tween-20) until excess Ponceau solution was removed, and imaged for loading control analysis, or with 1:5000 anti-Actin Antibody (Agrisera, AS13 2640). The membrane was blocked in 8% milk (Great Value) in TBS-T buffer for 1 hour. 1:5000 Anti-GFP (Agrisera, AS20 4443) or 1:5000 Anti-ARF7 antibody was added overnight at 4°C. The membrane was then washed 3 times in TBS-T solution for 5min before being incubated with anti-rabbit secondary antibody at 4°C overnight. The signal was detected using a WesternBright ECL HRP substrate kit (Advasta) according to manufacturer’s instructions.

For temperature-dependent assays, 4-day old Arabidopsis tissue was placed in water pre-warmed to the treatment temperature for 4 hours (unless otherwise stated). After 4 hours, seedlings were dabbed dry on a paper towel and flash frozen in liquid nitrogen and used in immunoblot analysis.

To examine the effects of the 26S proteasome on ARF stability, ARF levels in seedlings treated with the proteasomal inhibitor MG132 were examined. MG132 (Sigma-Aldrich) was dissolved in DMSO and supplemented to pre-warmed water to a final concentration of 50µM. For mock treatments, an equivalent amount of DMSO was supplemented to water. 30 4-day-old seedlings were then added to either treatment and incubated at the described temperature for four hours. After incubation, tissue was dabbed dry on a paper towel and flash frozen in liquid nitrogen for immunoblot analysis.

To examine the effects of the autophagy on ARF stability, ARF levels in seedlings treated with the autophagy inhibitor Concanamycin A were examined. Concanamycin A was dissolved in DMSO. Concanamycin A, Concavlin A, or the equivalent volume of DMSO, was then added to pre-warmed water to a final concentration of 1µM. Plants were left to incubate at the indicated temperature for 16h. After incubation, plants were dabbed dry on a paper towel and flash frozen in liquid nitrogen for immunoblot analysis.

To determine the half-lives of ARF proteins at different temperatures, seed lines were pre-treated at their prescribed temperature for one hour in a 6-well plate. Then, cycloheximide, dissolved in water, was added to each well and lightly mixed to ensure even diffusion of the chemical within the well. Plants were then collected from each well at the described time (immediately, 1h, 2h, or 4h later). Tissue was dabbed dry on a paper towel and flash frozen in liquid nitrogen for immunoblot analysis.

Protein Screen
An ARF19 mutagenesis library was created using a protocol adapted from Manel’s 2-plasmid Mutagenesis protocol25. The pUBQ10:YFP-ARF19c binary vector11 was transformed into competent JS200 cells carrying the EP pol I plasmid (Addgene). Overnight cultures were grown statically overnight at 30°C. The following morning, cultures were shaken for 2 hours at 180 rpm and 2µl of each culture was used to inoculate 5ml of 2xYT media with antibiotics. Mutagenesis was stimulated by placing cultures statically at 37°C for 15min and then shaken overnight at 37°C at 180 rpm. Cultures were miniprepped and transformed into JS200 cells and plated at 30°C in a serial dilution to approximate the number of transformation events present in each culture. All colonies on the plate were pooled together in LB. Cultures were then used to inoculate 5mL of 2XYT media and a second mutagenesis step was performed. The rate of mutagenesis was determined by transforming plasmids from the error-prone polymerase into NEB5a cells and sequencing. Mutagenized plasmids were then transformed into agrobacterium (GV3101). Agrobacterium colonies were then pooled and used to transform Col-0 plants using the floral dip method44. Positive transformants were selected using BASTA. Transformants were grown as individuals and their progeny were plated on a glass-bottom 24-well plate supplemented with PNS media. After 5 days of growth, seedlings were imaged when their roots grew along the bottom of the glass-bottom plate using a Leica DMI8 Thunder light microscope using an OKO Lab temperature-controlled stage at 32°C. Seedlings were imaged before and after heat treatment. Mutations in YFP-ARF19 were determined by performing a PCR reaction using primers present only in the binary vector backbone (5′-CAACAATTACCAACAACAAC-3′ and 5′-ACCTAGGATCATCAACC-3′) to prevent amplification of the endogenous ARF19 gene and sequenced.

Protoplast generation and experiments
Arabidopsis mesophyll protoplasts were isolated from 12 to 14-day-old Col-0 leaves using the tape sandwich method45. Approximately 100,000 cells were transformed with 20ug of plasmid containing pUBQ10:mNEON-ARF19-P2A-mScarlet3, or variants in which ARF19 was replaced by an ARF19 variant and incubated for 16 hours in the dark. Following incubation, protoplasts were either left at ambient temperature or treated at 32°C for four hours. The Beckman Coulter Cytoflex S Flow Cytometer was used to score the transformed protoplasts. Intact protoplasts were identified with a backgating strategy. mNEON levels were collected using the B525 channel (Ex: 488nm Em: 525±40nm, 69 gain). mScarlet levels were collected using the Y610 channel (Ex: 561nm Em: 610±20nm, 1000 gain). FCS files were generated using CytExpert software and analyzed with FlowKit46, using NumPy47 and Pandas48 packages in Python as previously described. Stability was calculated by dividing the mNEON signal by the mScarlet signal for each cell. Graphs were generated with seaborn49 and Matplotlib50 in Python.

Microscopy
All images were acquired using a Leica DMi8 Fluorescence microscope. Nuclei of seedlings carrying the pARF7:mVenus-ARF7 or pUBQ10:YFP-ARF19 reporter were imaged using a Leica DMi8 Confocal microscope. Nuclei were measured in 4-day-old seedlings in young epidermal cells with or without a four-hour treatment at 37°C. Nuclei fluorescence intensity was quantified using ImageJ Software.

For time course images depicting pUBQ10:YFP-ARF19 thermal accumulation, 4-day-old seedlings were mounted in water and imaged on a Leica DMI8 Thunder light microscope using an OKO Lab temperature-controlled stage at 32°C and imaged approximately every 12min over the course of 4 hours.

Natural Variation Assay
Fifteen Arabidopsis accessions were selected for their geographic diversity. To determine thermomorphogenic hypocotyl elongation, seedlings were grown with continuous illumination under yellow filters for 6 days at either 22°C or 28°C and hypocotyls were measured using FIJI. To conduct western blot analysis, 30 seedlings of each genotype were placed in pre-warmed water at 22°C or 37°C and incubated for four hours prior to protein extraction. Relative ARF7 protein levels were measured using FIJI by quantifying signal within the 37°C band and dividing by the 22°C band.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In their study, Wilkinson et al. argue that ARF7 and ARF19 function as thermosensors independent of both auxin biosynthesis and the canonical phyB-PIF4 signaling pathway. While I acknowledge some of the biochemical data, such as the reported increase in ARF protein levels at elevated temperatures, I disagree with the authors' interpretation of several blots. More importantly, I challenge two central aspects of their model: the assertion that this thermosensitive behavior operates independently of auxin biosynthesis and independently of the canonical phyB signaling pathway. Furthermore, I did not observe any convincing phenotypes that would support the biological relevance of this proposed mechanism.\nIn summary, while the authors interesting biochemical data, they fall short in establishing a causal link to thermosensitive growth phenotypes. What remains is an intriguing observation of thermosensitive behavior in two proteins previously known to be involved in canonical thermosignaling. Finally, it would be beneficial if the authors avoided speculative interpretations and ensured their conclusions were supported by appropriate statistical testing.\nGeneral remarks to the presented growth phenotypes\nHypocotyl elongation is the classic indicator for assessing thermomorphogenic responses. The phenotypes presented for the arf7arf19 double mutants are weak, suggesting that while ARF7 and ARF19 may have the ability to sense temperature changes, their biological relevance in this process appears either masked by functional redundancy with other ARFs or negligible. If the latter is true, the impact of the findings, despite being biochemically intriguing, would be moderate, indicating that a more specialized journal might be more suitable for publication.\nAdditionally, a key experiment in the study, which purportedly demonstrates the uncoupling of arf7/19 phenotypes from auxin biosynthesis (the same applies to the proposed independence from phyB signaling), lacks statistically robust data to support this conclusion. As a result, it remains unclear whether the demonstrated thermosensory behavior of ARF7/19 proteins is causally linked to the weak phenotypes reported. In my view, a purely correlative relationship cannot be excluded and would need to be addressed to establish the biological relevance of their thermosensory behavior.\nTaken together, this suggests that a) the phenotypes observed in arf7/19 mutants and b) their supposed independence from auxin biosynthesis are not convincingly demonstrated. Consequently, the study may ultimately be showing an effect of temperature-induced auxin biosynthesis followed by canonical auxin signaling mediated by ARF7 and ARF19, making it more confirmatory than novel in the context of thermomorphogenesis signaling.\nSpecific comments\nLine 38: Gangappa et al. does not seem to be the appropriate reference here. The first paper that showed the transpirative cooling effect was Crawford et al. (2012, Current Biology).\nFig. 1A-C:\n- Providing only relative data quantifications can be misleading. To assess the biological significance of the findings in this study, it is essential to report absolute growth data for each temperature tested. This allows the reader to determine whether the arf7arf19 double mutants behave like WT at control temperatures—implying a conditional phenotype at higher temperatures—or if the results point to a broader developmental issue. This clarity is especially important given that the relative growth phenotypes in arf7arf19 mutants are not particularly pronounced (in other words rather weak), especially when compared to the loss-of-function phenotypes of other thermosensors, such as phyB and elf3 mutants. This interpretation is supported by the data provided for growth at 22°C and 28°C in arf7arf19 mutants in Fig. S1C), which a) indicate that arf7arf19 double mutants display a very solid temperature response, and b) that arf7arf19 hypocotyls are already longer at 22°C (if the authors would have applied statistics, this might be more clear). This suggests that the observed phenotypes may be due to a general developmental defect rather than a specific temperature response.\n- In summary, the relatively weak growth phenotypes observed suggest that these findings may represent a secondary layer of thermosensory responses, which nonetheless appears to influence seedling behavior. However, the growth data presented in Fig. S1C raise significant questions about the biological relevance of these phenotypes.\n- Please describe which statistical test has has been performed.\nFig. S1B + line 65: I am somewhat surprised by the lack of response of the WT regarding the cotyledon petiole angle. As this basically serves as a proxy for hyponasty, it means that there was no response in the WT. This in turn means that the assay did not work, doesn’t it?\nFig. 1E + Fig. S1C,D + line 71: If the thermomorphogenic response is dampened, then I expect a statistically significant effect to be reported. However, no statistics has been performed. Please, do so.\nLines 74-79 + Fig. S1C: This experiment is critical, as its results could potentially decouple the effects of ARF7/19 on thermomorphogenesis from auxin biosynthesis. The authors state, \"Inclusion of auxin biosynthesis inhibitors did not fully block thermomorphogenic hypocotyl elongation in either wild type or in the 35S line (Fig. 1h,i, S1c,d). Conversely, inclusion of auxin biosynthesis inhibitors diminished arf7arf19 hypocotyl thermomorphogenesis (Fig. 1h,i, S1c,d).\" The phrasing here is suggestive, implying that WT and 35S are “not fully blocked,” while arf7/19 are “diminished,” suggesting a qualitative difference in response. However, this distinction cannot be properly evaluated due to the absence of statistical analysis, making it impossible to determine if arf7/19 mutants still show a response. If they do, they too would be classified as “not fully blocked.” Conversely, Fig. S1C indicates that the WT may also exhibit a diminished response, similar to arf7/19. Overall, these data do not conclusively demonstrate an uncoupling of arf7/19 phenotypes from auxin biosynthesis, contrary to the authors' claim that “Our data suggest that thermomorphogenesis partially depends on ARF7 and ARF19, independent of temperature-regulated auxin biosynthesis.” The observed phenotypes could still be attributed to elevated auxin levels and canonical TIR1/AFB signaling.\nLine 88: Please check reference to Fig. 1I\nFig. 1I: It should be possible to quantify the fluorescent signals.\nFig. 1M: When the antibody detects both ARFs, the signal you are detecting can come from either both or only one of the two proteins, correct? Or can you distinguish this? And why does the figure say ARF7 only, but the text refers to both proteins (lines 95-97)?\nFig. 2C,D: While the figure legend of 2C describes root tip nuclei, the legend for 2D does not mention which tissue was sampled. Why 22 vs. 28°C in D and 22 vs. 37°C in C? Furthermore, please refer to 2C and 2D also in the text.\nLines 131-135: The authors state, \"In time-course imaging of hypocotyls exposed to elevated temperature, we observed a rapid increase in nuclear signal (8 minutes post-exposure) prior to the occurrence of cytoplasmic condensates (~31 minutes post-exposure), congruent with a model in which elevated temperature conditions allow for increases in soluble/nuclear ARF protein.\" Please reference the specific data or figure corresponding to this description in the text. If the authors are referring to Fig. 1E, I am unable to discern this rapid increase from the figure. Additionally, including some form of quantification would help readers evaluate the authors' interpretation without relying solely on representative images.\nLine 159: The first papers that showed the involvement of HY5 in temperature responsive processes were Toledo-Ortiz et al., (2014, PloS Genetics) and Delker et al. (2014, Cell Reports. Gangappa et al. (2017, Cell Reports) then showed that HY5 competes with PIF4 for transcription factor binding sites. These references might be better suited than Lee et al. (2021).\nLine 160: Similarly, Box et al. (2015, Current Biology) and Raschke et al. (2015, BMC Plant Biology) demonstrated the temperature-dependent interaction between ELF3 and PIF4 in the same year as Nieto et al. (2015). The thermosensory properties of this relationship were then uncovered by Jung et al. (2020, Nature).\nLine 161: In this paragraph, original research papers are cited for all other signaling genes, so it is inconsistent to reference a review for phyB. To maintain consistency, consider citing the original research articles (e.g., Jung et al., 2020; Legris et al., 2020, both published in Nature). Alternatively, if a review is preferred for uniformity, cite a comprehensive review that covers all relevant aspects, such as Casal et al. (2019).\nFig. 2J: The authors claim that ARF7/19 protein levels remain unaffected in a hy5 mutant background. Based on the blot provided, I must respectfully disagree. It is evident that at 32°C, ARF7 is more stable in the hy5 background compared to the wild type. This finding strongly supports the idea that ARF7 operates via the canonical pathway (and not independently), as HY5 functions as a negative regulator. In the absence of HY5, PIF4 would be free to induce auxin biosynthesis through YUC8, potentially stabilizing ARF proteins. If these blots are indeed representative, then I believe the authors have misinterpreted their results. In fact, the data from the hy5 mutant would suggest the opposite conclusion: that ARF7/19 depend on the canonical thermomorphogenesis signaling pathway.\nFig. 2K: I believe it would be unproductive to perform this experiment with a phyB/pifQ mutant. phyB and PIFs act in opposite directions: phyB loss-of-function mutants exhibit elongated hypocotyls at all temperatures, alongside various other defects, while pifQ mutants are largely unresponsive to temperature and remain short. Although I have not encountered a combination of both mutations, based on their genetic interactions, they should behave similarly to pifQ alone. Therefore, it would make more sense to separate these mutant lines for clarity and to assess their individual contributions.\nFig. 2L: I go along with the suggestion that ARF7 overaccumulates in the 35S:ELF3 line at 22°C control temperature. But the 32°C and 37°C bands require quite some creativity to safely call these more intense than the WT bands. Therefore, either provide robust quantification to support this claim or refrain from drawing conclusions based on unclear data—especially since the actin control band is also more intense at 32°C in the 35S line.\nLines 174-175: In response to my above mentioned criticism of the data interpretation presented in Fig. 2J-L, I must disagree with the assertion that the accumulation of ARF proteins at elevated temperatures is independent of the canonical temperature signaling pathway.\nFig. 3B + lines 178-185: How do the authors explain that MR+PB1 becomes instable at high temperature, which the DBD is stabilized by the same conditions?\nFig. 3E: The authors claim that ARF19W330L loses its increased stability at high temperature. While I find the 3D fluorescent images, lacking quantification, insufficient to support or critique this claim, I do observe a clear increase in the ARF19W330L protein band at high temperature, despite the actin control band being only slightly more intense. Why, then, do the authors interpret this in the opposite way? As for the other allele, it appears there is no detectable signal at either temperature. Could it be that this allele exhibits a general stability defect, independent of temperature?\nFig. 3G: Statistics?\nFig. 4B + line 217: Ler ARF levels are supposedly slightly increased at high temperature. However, the figure displays a high temperature band intensity of 0.9. In my understanding indicates a decrease rather than an increase.\nLine 219: Shouldn’t this be 4B instead of 4C?", "reviewer_2": "In their manuscript, Wilkinson et al. report that AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 contribute to the plant’s temperature response not only by interpreting increased auxin levels, but also by showing temperature responsiveness themselves: elevated temperature increases stability of the two proteins and increases the concentration of soluble ARF7/19 in the nucleus as opposed to accumulation in inactive cytoplasmic condensates. ARF7/19 temperature responsiveness persists in many known temperature signalling mutants. Finally, ARF7/19 accumulation correlated with the temperature response across a set of 14 Arabidopsis accessions, suggesting it has contributed to local temperature adaptation.\nUnderstanding temperature perception and signalling in plants has attracted much attention in recent years. A novel temperature response pathway should be of considerable interest to the plant science community and beyond. I have some recommendations for further improving the manuscript (details below), but the findings presented, and their interpretation, are sound.\nMajor points\n1) A major point of the manuscript is that ARF7/19 integrate temperature information independently of auxin biosynthesis. To further strengthen this hypothesis, ARF7/19 levels should be analysed in a YUC overexpressor (which displays a constitutive warm temperature response) and a yucQ quadruple mutant (in which thermomorphogenesis is nearly abolished) using the custom ARF7/19 antibody.\n2) Wilkinson et al. find that two mutations (W330L and R297W) strongly affect thermostability of ARF7/19. Is condensate formation also altered by these mutations? This can be easily tested with the YFP-tagged ARF19-W330L and ARF19-R297W lines and would further clarify whether these mutations mainly affect protein stability or are also involved in oligomerisation/condensate formation.\nMinor points\n3) Fig 1e, S1c, S1d: Statistics are missing.\n4) Fig. S2: It is unclear to me which bands correspond to ARF7 and ARF19 (and the respective tagged versions) – this should be labelled. Also, mutant names should be lower case (arf7-1, arf19-1)\n5) Fig. 2a: What temperatures do concentrations c1 and c2 correspond to – 22 and 32 degrees Celsius?\n6) Fig. 2e: Larger images would be needed to appreciate the dynamics of ARF19 accumulation. I suggest pointing out nuclei versus cytoplasmic condensates with arrows or similar (as this is not inherently obvious).\n7) Fig. 2g: The legend mentions an n equal or greater than 1340. Is this correct?", "reviewer_3": "Elevated temperatures result in increased auxin biosynthesis, and ARF family mediated transcriptional responses to auxin are necessary for thermomorphogenesis. This study by Wilkinson et.al., aimed to confirm whether AUXIN RESPONSE FACTOR ARF7 and ARF19 can be directly affected by temperature independently of upstream auxin. They found that ARF proteins accumulate in response to elevated temperature due to its enhanced stability. It was also observed that temperature-regulated ARF7 and ARF19 nucleo-cytoplasmic partitioning in auxin response. Finally, they found that natural variation in thermoregulated ARF accumulation was correlated with thermomorphogenesis. The finding of this manuscript is interesting, but I do have several concerns about it.\nLine 72-79 The conclusion that role of ARF7/19 in thermomorphogenesis is independent of auxin biosynthesis was not well supported by current data. Inclusion of auxin biosynthesis inhibitors did not fully block thermomorphogenic hypocotyl elongation in either wild type or in the 35S:YUC1 but diminished arf7 arf19 hypocotyl thermomorphogenesis could result from the incomplete inhibition of auxin by the inhibitors. In this case, arf7 arf19 has additional effect with auxin inhibition. Also, other thermomorphogenesis pathways exist, so it is not surprising that auxin inhibitor cannot fully block thermomorphogenic hypocotyl elongation in wild type. The auxin levels in all genotypes under different temperatures in the absence or presence of auxin inhibitors should be quantified.\nA key point of this manuscript is that temperature regulates ARF7 and ARF19 solubility. However, this is not well-supported. This should be tested in different systems such as in vitro or in heterologous expression systems.\nLine 145-152 The authors indicate an increase in stability and solubility of ARF protein with temperature, yet the experiments were not designed to separately verify the independent effects of temperature on stability and solubility. Due to the combined effects of the two, it becomes challenging to discern the precise mechanism by which temperature alters the accumulation and distribution of ARF protein. I don't understand the relationship between the solubility, stability and nuc/cyto portioning of ARF7 and ARF19, do they have causal relationship? A working model might be helpful for readers to comprehend.\nLine106-122 The authors have not provided a clear explanation for the cause of ARF thermal stability, and how ARF manages to evade degradation and maintain a certain protein level.\nLine 130-135 The image quality of figure 2b&2e are insufficient for me to extract any information. Is the phenomenon of temperature-induced protein nuclear localization and aggregation similar to previously reported ARF functions? Are these two phenomena induced by temperature spontaneous, or do they also rely on auxin or other signaling pathways? Is there a relationship between these phenomena and the protein's thermal stability? Please explain it.\nMutagenesis experiments suggest potential structural bases for ARF thermal stability, with two mutations in the DNA binding domain (DBD) influencing its thermal stability. It would be necessary to show thermomorphogenesis phenotypes in ARF19 W330L and ARF19 R297W variant. And, this explanation is based solely on predictive analysis and does not clarify how the critical sites contribute to the protein's acquisition of thermal stability. Perhaps it affects nuclear translocation or downstream signal transduction. Is it the case that the protein undergoes structural changes under heat, making it less susceptible to degradation?\nAs the protein level changes are the main molecular phenotype of this manuscript, all immunoblots should be performed at least three times and quantification of the intensity should be performed with error bars included.\nThe immunoblot experiments in Fig. 1m should be repeated with proper loading control.\nFig.2c-e were not referred in the manuscript.\nLine145, essential information is lacking to link to the statement “Our data is consistent with the possibility that ARF7 and 19 display upper critical solution 145 temperature (UCST) behavior”.\nLine76-77 Fig.1d,e rather than Fig.1h, i should be referred to.\nLine200-201, Fig.3f rather than Fig.3g should be referred to."}, "author_response": "We thank the reviewers for their constructive criticism. Their comments led us to perform\nadditional experiments (described below), which we believe has strengthened our claims and the\npotential impact of this study.\nReviewer #1 (Remarks to the Author):\nIn their study, Wilkinson et al. argue that ARF7 and ARF19 function as thermosensors\nindependent of both auxin biosynthesis and the canonical phyB-PIF4 signaling pathway.\nWhile I acknowledge some of the biochemical data, such as the reported increase in ARF\nprotein levels at elevated temperatures, I disagree with the authors' interpretation of\nseveral blots. More importantly, I challenge two central aspects of their model: the\nassertion that this thermosensitive behavior operates independently of auxin biosynthesis\nand independently of the canonical phyB signaling pathway. Furthermore, I did not\nobserve any convincing phenotypes that would support the biological relevance of this\nproposed mechanism.\nIn summary, while the authors interesting biochemical data, they fall short in establishing\na causal link to thermosensitive growth phenotypes. What remains is an intriguing\nobservation of thermosensitive behavior in two proteins previously known to be involved in\ncanonical thermosignaling. Finally, it would be beneficial if the authors avoided speculative\ninterpretations and ensured their conclusions were supported by appropriate statistical\ntesting.\nGeneral remarks to the presented growth phenotypes\nHypocotyl elongation is the classic indicator for assessing thermomorphogenic responses.\nThe phenotypes presented for the arf7arf19 double mutants are weak, suggesting that while\nARF7 and ARF19 may have the ability to sense temperature changes, their biological\nrelevance in this process appears either masked by functional redundancy with other ARFs\nor negligible. If the latter is true, the impact of the findings, despite being biochemically\nintriguing, would be moderate, indicating that a more specialized journal might be more\nsuitable for publication.\nIndeed, in our initial submission, we observed that the thermomorphogenesis phenotypes for arf7\narf19 are subtle, despite the dramatic effects of temperature on ARF7 and ARF19 accumulation.\nIn Arabidopsis, there are five Class-A (activator) ARFs, which are thought to positively drive\nauxin-responsive gene expression. ARF7 and 19 are closely-related, with ARF5 as the next\nmost-closely related to ARF7 and 19. ARF6 and 8 are additional Class-A ARFs in a separate\nclade from ARF7, 19, and 5. To determine whether the mismatch in the severity of arf7 arf19\nthermomorphogenesis phenotype and ARF7 and ARF19 thermoresponsive accumulation was\npotentially caused by functional redundancy, we examined an ARF5 reporter and found that,\nsimilar to ARF7 and ARF19, ARF5 undergoes a dramatic increase in accumulation in response\nto elevated temperature. Unfortunately, arf5 mutants are not viable and thus we are unable to\nassess the effects of loss of ARF5 on thermomorphogenesis. We have included this new data as\nFigure 2j and 2k.\nAdditionally, a key experiment in the study, which purportedly demonstrates the\nuncoupling of arf7/19 phenotypes from auxin biosynthesis (the same applies to the\nproposed independence from phyB signaling), lacks statistically robust data to support this\nconclusion. As a result, it remains unclear whether the demonstrated thermosensory\nbehavior of ARF7/19 proteins is causally linked to the weak phenotypes reported. In my\nview, a purely correlative relationship cannot be excluded and would need to be addressed\nto establish the biological relevance of their thermosensory behavior.\nTo address this concern, we performed rescue experiments of arf7 arf19 with our identified\nARF19 variants that fail to display thermoresponsive protein accumulation. In these experiments,\nwe find two ARF19 variants that both fail to demonstrate robust temperature-responsive protein\naccumulation and fail to fully rescue arf7 arf19 thermomorphogenic hypocotyl elongation. This\ndata is now included in Figure 5.\nTaken together, this suggests that a) the phenotypes observed in arf7/19 mutants and b)\ntheir supposed independence from auxin biosynthesis are not convincingly demonstrated.\nConsequently, the study may ultimately be showing an effect of temperature-induced auxin\nbiosynthesis followed by canonical auxin signaling mediated by ARF7 and ARF19, making\nit more confirmatory than novel in the context of thermomorphogenesis signaling.\nWe feel that the addition of data demonstrating that 1) an additional Class-A ARF displays\nthermoresponsive protein accumulation and thus may be functionally redundant with ARF7 and\nARF19, and 2) ARF19 variants that fail to display thermoresponsive protein accumulation fail to\nrescue arf7 arf19 thermomorphogenesis phenotypes strengthen our conclusions that temperature-\nresponsive ARF accumulation acts as a mechanism to integrate temperature cues and growth\nresponses.\nSpecific comments\nLine 38: Gangappa et al. does not seem to be the appropriate reference here. The first\npaper that showed the transpirative cooling effect was Crawford et al. (2012, Current\nBiology).\nThank you for catching this mistake. We now use the correct reference.\nFig. 1A-C:\n- Providing only relative data quantifications can be misleading. To assess the biological\nsignificance of the findings in this study, it is essential to report absolute growth data for\neach temperature tested. This allows the reader to determine whether the arf7arf19 double\nmutants behave like WT at control temperatures—implying a conditional phenotype at\nhigher temperatures—or if the results point to a broader developmental issue. This clarity\nis especially important given that the relative growth phenotypes in arf7arf19 mutants are\nnot particularly pronounced (in other words rather weak), especially when compared to\nthe loss-of-function phenotypes of other thermosensors, such as phyB and elf3 mutants.\nThis interpretation is supported by the data provided for growth at 22°C and 28°C in\narf7arf19 mutants in Fig. S1C), which a) indicate that arf7arf19 double mutants display a\nvery solid temperature response, and b) that arf7arf19 hypocotyls are already longer at\n22°C (if the authors would have applied statistics, this might be more clear). This suggests\nthat the observed phenotypes may be due to a general developmental defect rather than a\nspecific temperature response.\nWhereas indeed, the arf7 arf19 double mutant displays developmental defects, they do display\ndecreased thermomorphogenesis. The only way to assess this is by comparing the growth\nchanges at different temperatures, rather than simply absolute final sizes. As noted, we include\nabsolute lengths in Figure 1 for the sake of data transparency. However, to interrogate this\nphenotype more closely, we examined cellular growth rates in distinct zones of the hypocotyl,\nfinding that the cells most responsive to temperature change in wild type are in the middle zone.\nThese cells ‘start off’ longer in arf7 arf19 and do not display a robust increase in length in\nresponse to elevated temperature. We now include this data as Figures 1d and 1e. Additionally,\nwe examine thermoresponsive adult leaf angle changes, finding a defect in this process. This data\nhas been included in Figure 1f.\n- In summary, the relatively weak growth phenotypes observed suggest that these findings\nmay represent a secondary layer of thermosensory responses, which nonetheless appears to\ninfluence seedling behavior. However, the growth data presented in Fig. S1C raise\nsignificant questions about the biological relevance of these phenotypes.\n- Please describe which statistical test has been performed.\nWe have performed statistical analyses throughout the work; these statistics support our\nconclusions. The performed statistics are described in each figure legend. In addition, we have\nperformed more careful analysis of arf7 arf19 thermomorphogenesis, in collaboration with the\nlab of Jorge Casal, to gain an understanding of the region of the hypocotyl that displays reduced\nthermomorphogenic growth. These new data are included in Figure 1.\nFig. S1B + line 65: I am somewhat surprised by the lack of response of the WT regarding\nthe cotyledon petiole angle. As this basically serves as a proxy for hyponasty, it means that\nthere was no response in the WT. This in turn means that the assay did not work, doesn’t\nit?\nWe agree. Because this data did not add to our overall model, we have removed it from our\nmanuscript.\nFig. 1E + Fig. S1C,D + line 71: If the thermomorphogenic response is dampened, then I expect a\nstatistically significant effect to be reported. However, no statistics has been performed. Please,\ndo so.\nWe have now performed statistical analyses, as requested.\nLines 74-79 + Fig. S1C: This experiment is critical, as its results could potentially decouple\nthe effects of ARF7/19 on thermomorphogenesis from auxin biosynthesis. The authors\nstate, \"Inclusion of auxin biosynthesis inhibitors did not fully block thermomorphogenic\nhypocotyl elongation in either wild type or in the 35S line (Fig. 1h,i, S1c,d). Conversely,\ninclusion of auxin biosynthesis inhibitors diminished arf7arf19 hypocotyl\nthermomorphogenesis (Fig. 1h,i, S1c,d).\" The phrasing here is suggestive, implying that\nWT and 35S are “not fully blocked,” while arf7/19 are “diminished,” suggesting a\nqualitative difference in response. However, this distinction cannot be properly evaluated\ndue to the absence of statistical analysis, making it impossible to determine if arf7/19\nmutants still show a response. If they do, they too would be classified as “not fully\nblocked.” Conversely, Fig. S1C indicates that the WT may also exhibit a diminished\nresponse, similar to arf7/19. Overall, these data do not conclusively demonstrate an\n demonstrate an\nuncoupling of arf7/19 phenotypes from auxin biosynthesis, contrary to the authors' claim\nthat “Our data suggest that thermomorphogenesis partially depends on ARF7 and ARF19,\nindependent of temperature-regulated auxin biosynthesis.” The observed phenotypes could\nstill be attributed to elevated auxin levels and canonical TIR1/AFB signaling.\nTo confirm that auxin biosynthesis was indeed blocked with our treatments, we used the DII-\nVENUS reporter (Brunoud et al, 2012) to assay the effect of our treatments on auxin levels. We\nfound that DII-VENUS signal decreased with elevated temperature in the absence of inhibitor,\nconsistent with elevated auxin biosynthesis. In contrast, there is no difference in DII-VENUS\nsignal at 22C vs 28C when auxin biosynthesis inhibitors are included. These data suggest that\nour inhibitors are effective at blocking auxin biosynthesis in these assays. This data is now\nincluded in Figure 1.\nLine 88: Please check reference to Fig. 1I\nWe have double-checked this reference.\nFig. 1I: It should be possible to quantify the fluorescent signals.\nWe have included a quantification of fluorescence levels of UBQ10:ARF19-YFP from Figure 2e\nas Figure 2f (the figure numbers have changed in the revision).\nFig. 1M: When the antibody detects both ARFs, the signal you are detecting can come from\neither both or only one of the two proteins, correct? Or can you distinguish this? And why\ndoes the figure say ARF7 only, but the text refers to both proteins (lines 95-97)?\nThe custom antibody was raised against the PB1 domain of ARF7, but has cross-reactivity to\nARF19, which we report in Supplemental Figure 2 (now better annotated). Because the signal\nfrom ARF19 is often faint in comparison to ARF7 (as might be expected, as the antibody was not\nraised to ARF19), we are not consistently able to detect the ARF19 cross-reacting band. For this\nreason, we crop our images to only view ARF7, as ARF19 is quite faint in most of our blots.\nFig. 2C,D: While the figure legend of 2C describes root tip nuclei, the legend for 2D does\nnot mention which tissue was sampled. Why 22 vs. 28°C in D and 22 vs. 37°C in C?\nFurthermore, please refer to 2C and 2D also in the text.\nThe tissue in figure 2D also refers to root tips. Figures 2C and 2E are now referenced to in the\ntext. Because the plant response to elevated temperature is different with temperatures that\npromote thermomorphogenesis (28C) and heat shock (37C), we wanted to determine the effects\nat both of these temperatures.\nLines 131-135: The authors state, \"In time-course imaging of hypocotyls exposed to\nelevated temperature, we observed a rapid increase in nuclear signal (8 minutes post-\nexposure) prior to the occurrence of cytoplasmic condensates (~31 minutes post-exposure),\ncongruent with a model in which elevated temperature conditions allow for increases in\nsoluble/nuclear ARF protein.\" Please reference the specific data or figure corresponding to\nthis description in the text. If the authors are referring to Fig. 1E, I am unable to discern\nthis rapid increase from the figure. Additionally, including some form of quantification\nwould help readers evaluate the authors' interpretation without relying solely on\nrepresentative images.\nTo visualize this data better, we now provided larger micrographs of this result, as well as\nquantification of nuclear signal at 0 and 31 minutes. This is provided in Figure 3.\nLine 159: The first papers that showed the involvement of HY5 in temperature responsive\nprocesses were Toledo-Ortiz et al., (2014, PloS Genetics) and Delker et al. (2014, Cell\nReports. Gangappa et al. (2017, Cell Reports) then showed that HY5 competes with PIF4\nfor transcription factor binding sites. These references might be better suited than Lee et\nal. (2021).\nThank you for pointing out this mistake. We have now corrected the citations.\nLine 160: Similarly, Box et al. (2015, Current Biology) and Raschke et al. (2015, BMC\nPlant Biology) demonstrated the temperature-dependent interaction between ELF3 and\nPIF4 in the same year as Nieto et al. (2015). The thermosensory properties of this\nrelationship were then uncovered by Jung et al. (2020, Nature).\nThank you for pointing out this mistake. We have now corrected the citations.\nLine 161: In this paragraph, original research papers are cited for all other signaling genes,\nso it is inconsistent to reference a review for phyB. To maintain consistency, consider citing\nthe original research articles (e.g., Jung et al., 2020; Legris et al., 2020, both published in\nNature). Alternatively, if a review is preferred for uniformity, cite a comprehensive review\nthat covers all relevant aspects, such as Casal et al. (2019).\nThank you for pointing out this inconsistency. We have now cited primary literature for phyB\nand the Casal review at the end to point readers to the correct references.\nFig. 2J: The authors claim that ARF7/19 protein levels remain unaffected in a hy5 mutant\nbackground. Based on the blot provided, I must respectfully disagree. It is evident that at\n32°C, ARF7 is more stable in the hy5 background compared to the wild type. This finding\nstrongly supports the idea that ARF7 operates via the canonical pathway (and not\nindependently), as HY5 functions as a negative regulator. In the absence of HY5, PIF4\nwould be free to induce auxin biosynthesis through YUC8, potentially stabilizing ARF\nproteins. If these blots are indeed representative, then I believe the authors have\nmisinterpreted their results. In fact, the data from the hy5 mutant would suggest the\nopposite conclusion: that ARF7/19 depend on the canonical thermomorphogenesis\nsignaling pathway.\nWe now show a more representative blot and quantification of several immunoblots that\ndemonstrate that hy5 displays normal temperature regulation of ARF7 accumulation.\nFig. 2K: I believe it would be unproductive to perform this experiment with a phyB/pifQ\nmutant. phyB and PIFs act in opposite directions: phyB loss-of-function mutants exhibit\nelongated hypocotyls at all temperatures, alongside various other defects, while pifQ\nmutants are largely unresponsive to temperature and remain short. Although I have not\nencountered a combination of both mutations, based on their genetic interactions, they\nshould behave similarly to pifQ alone. Therefore, it would make more sense to separate\nthese mutant lines for clarity and to assess their individual contributions.\nWe have now assessed the temperature-regulated accumulation of ARF7 in the phyB and pifQ\nmutant backgrounds, finding that ARF7 accumulation increases at elevated temperature in both\nof these. This data is included in Figure 3.\nFig. 2L: I go along with the suggestion that ARF7 overaccumulates in the 35S:ELF3 line at\n22°C control temperature. But the 32°C and 37°C bands require quite some creativity to\nsafely call these more intense than the WT bands. Therefore, either provide robust\nquantification to support this claim or refrain from drawing conclusions based on unclear\ndata—especially since the actin control band is also more intense at 32°C in the 35S\nline.\nWe now provide quantitative data for the 35S:ELF3 line. Given the range of protein levels, it is\ndifficult to consistently get exposures that adequately capture the range of protein accumulation,\nbut we have reported the data that we have for readers to judge for themselves. We have softened\nclaims around ELF3.\nLines 174-175: In response to my above mentioned criticism of the data interpretation presented\nin Fig. 2J-L, I must disagree with the assertion that the accumulation of ARF proteins at elevated\ntemperatures is independent of the canonical temperature signaling pathway.\nWe consistently see no major defects in ARF7 temperature-regulated accumulation in the pifQ\nand phyB backgrounds, supporting our claim that this is likely not regulated through the\ncanonical pathways.\nFig. 3B + lines 178-185: How do the authors explain that MR+PB1 becomes instable at high temperature, which the DBD is stabilized by the same conditions?\nBoth MR+PB1 and DBD display increased accumulation at elevated temperature. There is an\nunfortunate cross-reacting band (marked with an asterisk) that displays decreased stability at\nelevated temperature. We have made this more clear in the legend.\nFig. 3E: The authors claim that ARF19W330L loses its increased stability at high temperature.\nWhile I find the 3D fluorescent images, lacking quantification, insufficient to support or critique\nthis claim, I do observe a clear increase in the ARF19W330L protein band at high temperature,\ndespite the actin control band being only slightly more intense. Why, then, do the authors\ninterpret this in the opposite way? As for the other allele, it appears there is no detectable signal\nat either temperature. Could it be that this allele exhibits a general stability defect, independent\nof temperature?\nIn our initial submission, we characterized the primary transformants used in the screen, which\ndisplayed varying levels of ARF19 expression. To address this concern, we created new stable\nlines of the ARF19 variants in the arf7 arf19 mutant background so that we could assess their\nthermoresponsive accumulation and ability to rescue arf7 arf19 thermomorphogenesis. We find\nthat stable transformants of these variants display decreased thermoresponsive protein stability,\nsimilar to what we found when we previously tested these variants in a protein stability reporter\nin protoplasts. Further, these variants were unable to rescue arf7 arf19 thermomorphogenesis,\nconsistent with a model in which temperature-regulated ARF7 and 19 accumulation are\nnecessary for their full contribution to temperature response.\nFig. 3G: Statistics?\nThe statistics were accidentally deleted from the final version of the figure, it is now corrected.\nFig. 4B + line 217: Ler ARF levels are supposedly slightly increased at high temperature.\nHowever, the figure displays a high temperature band intensity of 0.9. In my\nunderstanding indicates a decrease rather than an increase.\nThank you for pointing this out, we have reworded the text accordingly.\nLine 219: Shouldn’t this be 4B instead of 4C?\nWe have corrected this figure callout.\nReviewer #2 (Remarks to the Author):\nIn their manuscript, Wilkinson et al. report that AUXIN RESPONSE FACTOR 7 (ARF7)\nand ARF19 contribute to the plant’s temperature response not only by interpreting\nincreased auxin levels, but also by showing temperature responsiveness themselves:\nelevated temperature increases stability of the two proteins and increases the concentration\nof soluble ARF7/19 in the nucleus as opposed to accumulation in inactive cytoplasmic\ncondensates. ARF7/19 temperature responsiveness persists in many known temperature\nsignalling mutants. Finally, ARF7/19 accumulation correlated with the temperature\nresponse across a set of 14 Arabidopsis accessions, suggesting it has contributed to local\ntemperature adaptation.\nUnderstanding temperature perception and signalling in plants has attracted much\nattention in recent years. A novel temperature response pathway should be of considerable\ninterest to the plant science community and beyond. I have some recommendations for\nfurther improving the manuscript (details below), but the findings presented, and their\ninterpretation, are sound.\nThank you for your support and comments for improving our manuscript.\nMajor points\n1) A major point of the manuscript is that ARF7/19 integrate temperature information\nindependently of auxin biosynthesis. To further strengthen this hypothesis, ARF7/19 levels\nshould be analysed in a YUC overexpressor (which displays a constitutive warm\ntemperature response) and a yucQ quadruple mutant (in which thermomorphogenesis is\nnearly abolished) using the custom ARF7/19 antibody.\nWe thank the reviewer for suggesting this experiment. We have found that altering auxin\nbiosynthesis does not affect ARF7 thermoresponsive accumulation in seedlings (Figure S5). We\nhave assayed ARF7 levels at 22°C and 32°C using our custom antibody in plants over expressing\nYUCCA1 (35s:YUCCA1) and reduced in the yucQ mutant (YUC3,5,7,8, and 9) and found that\nprotein levels of ARF7 in response to increased temperature are not drastically altered from wild\ntype.\n2) Wilkinson et al. find that two mutations (W330L and R297W) strongly affect\nthermostability of ARF7/19. Is condensate formation also altered by these mutations?\nThis can be easily tested with the YFP-tagged ARF19-W330L and ARF19-R297W lines and\nwould further clarify whether these mutations mainly affect protein stability or are also\ninvolved in oligomerisation/condensate formation.\nGiven the tight revision timeline, we were unable to collect this data in stable transformants.\nMinor points\n3) Fig 1e, S1c, S1d: Statistics are missing.\nStatistical analysis has now been added to these figures.\n4) Fig. S2: It is unclear to me which bands correspond to ARF7 and ARF19 (and the\nrespective tagged versions) – this should be labelled. Also, mutant names should be lower\ncase (arf7-1, arf19-1)\nWe have now labeled the bands and have made the genotypes of each lane more clear.\n5) Fig. 2a: What temperatures do concentrations c1 and c2 correspond to – 22 and 32\ndegrees Celsius?\nThis is a standard upper critical saturation temperature (UCST) phase diagram and is meant to\ngraphically represent how, in these systems, saturation concentration goes up with increasing\ntemperature (ie, the protein becomes more soluble). It is not meant to explicitly represent specific\nconcentrations and temperatures for the ARF proteins. Determining the full phase diagram of\nthese proteins is not technically feasible at this time. We have provided a fuller description of the\nphase diagram in the legend.\n6) Fig. 2e: Larger images would be needed to appreciate the dynamics of ARF19 accumulation. I\nsuggest pointing out nuclei versus cytoplasmic condensates with arrows or similar (as this is not\ninherently obvious).\nWe have enlarged this image and pointed out nuclei and condensates as requested.\n7) Fig. 2g: The legend mentions an n equal or greater than 1340. Is this correct?\nYes, this is correct.\nReviewer #3 (Remarks to the Author):\nElevated temperatures result in increased auxin biosynthesis, and ARF family mediated\ntranscriptional responses to auxin are necessary for thermomorphogenesis. This study by\nWilkinson et.al., aimed to confirm whether AUXIN RESPONSE FACTOR ARF7 and\nARF19 can be directly affected by temperature independently of upstream auxin. They\nfound that ARF proteins accumulate in response to elevated temperature due to its\nenhanced stability. It was also observed that temperature-regulated ARF7 and ARF19\nnucleo-cytoplasmic partitioning in auxin response. Finally, they found that natural\nvariation in thermoregulated ARF accumulation was correlated with\nthermomorphogenesis. The finding of this manuscript is interesting, but I do have several\nconcerns about it.\nLine 72-79 The conclusion that role of ARF7/19 in thermomorphogenesis is independent of\nauxin biosynthesis was not well supported by current data. Inclusion of auxin biosynthesis\ninhibitors did not fully block thermomorphogenic hypocotyl elongation in either wild type\nor in the 35S:YUC1 but diminished arf7 arf19 hypocotyl thermomorphogenesis could\nresult from the incomplete inhibition of auxin by the inhibitors. In this case, arf7 arf19 has\nadditional effect with auxin inhibition. Also, other thermomorphogenesis pathways exist, so\nit is not surprising that auxin inhibitor cannot fully block thermomorphogenic hypocotyl\nelongation in wild type. The auxin levels in all genotypes under different temperatures in\nthe absence or presence of auxin inhibitors should be quantified.\nUnfortunately, we do not have facilities to measure auxin levels at Duke University. Thus, to\nconfirm that auxin biosynthesis was indeed blocked with our treatments, we used the DII-\nVENUS reporter (Brunoud et al, 2012) to assay the effect of our treatments on auxin levels. We\nfound that DII-VENUS signal decreased with elevated temperature in the absence of inhibitor,\nconsistent with elevated auxin biosynthesis. In contrast, there is no difference in DII-VENUS\nsignal at 22C vs 28C when auxin biosynthesis inhibitors are included. These data suggest that\nour inhibitors are effective at blocking auxin biosynthesis in these assays.\nTo further assess the effects of auxin levels on ARF7 accumulation, we assayed ARF7 levels at\n22°C and 32°C using our custom antibody in plants over expressing YUCCA1 (35s:YUCCA1)\nand reduced in the yucQ mutant (YUC3,5,7,8, and 9) and found that protein levels of ARF7 in\nresponse to increased temperature are not drastically altered from wild type.\nA key point of this manuscript is that temperature regulates ARF7 and ARF19 solubility.\nHowever, this is not well-supported. This should be tested in different systems such as in\nvitro or in heterologous expression systems.\nWe have tried for many years to express ARF7 and ARF19 heterologously to perform these\ntypes of assays in vitro. Despite our great efforts, we have been unable to express these proteins\nin their full length. In the companion article (Borniego et al), the authors express ARF proteins in\nyeast and examine solubility under different temperatures, finding that ARF7 and 19 are more\nsoluble at elevated temperature. We now cite this result in our manuscript.\nLine 145-152 The authors indicate an increase in stability and solubility of ARF protein with\ntemperature, yet the experiments were not designed to separately verify the independent effects\nof temperature on stability and solubility. Due to the combined effects of the two, it becomes\nchallenging to discern the precise mechanism by which temperature alters the accumulation and\ndistribution of ARF protein. I don't understand the relationship between the solubility, stability\nand nuc/cyto portioning of ARF7 and ARF19, do they have causal relationship? A working\nmodel might be helpful for readers to comprehend.\nWe have expanded our explanation of the relationship between protein concentration, condensate\nformation, and solubility. We hope that this has made this data more accessible to most readers.\nLine106-122 The authors have not provided a clear explanation for the cause of ARF\nthermal stability, and how ARF manages to evade degradation and maintain a certain\nprotein level.\nThis is a long-term interest of the lab. Indeed, we have now extended our examination of the\nnatural variation overlying temperature-regulated ARF accumulation to perform a genome-wide\nassociation study. In our GWAS, we identified several loci that regulate ARF temperature-\nresponsive stability, which will be the topic of follow-up studies, as we have identified loci that\nregulate thermoresponsive ARF protein accumulation. Although these genes have not previously\nbeen implicated in temperature response, mutants defective in the identified loci fail to\ndemonstrate temperature-responsive ARF accumulation and also display defects in\nthermomorphogenesis that exceed those displayed by the arf7 arf19 mutant, suggesting that this\nhas been an effective method to identify regulators of temperature response. Although we have\nnot yet gotten to the point in the study of these newly identified factors to have a molecular\nmechanism underlying this process, we are actively working toward this (it will be ~2 years\nbefore this project is complete).\nLine 130-135 The image quality of figure 2b&2e are insufficient for me to extract any\ninformation. Is the phenomenon of temperature-induced protein nuclear localization and\naggregation similar to previously reported ARF functions? Are these two phenomena\ninduced by temperature spontaneous, or do they also rely on auxin or other signaling\npathways? Is there a relationship between these phenomena and the protein's thermal\nstability? Please explain it.\nThank you for this critique – we have now created enlarged versions of these images that we\nbelieve are easier to see detail.\nIn addition, we go into more detail in the text on the relationship between temperature-reguated\nprotein accumulation and temperature-regulated protein solubility. Temperature-driven protein\nsolubility is a spontaneous process, similar to how many other molecules are more soluble in\nwarm solvents. The effects on protein accumulation are via an unknown mechanism.\nMutagenesis experiments suggest potential structural bases for ARF thermal stability, with two\nmutations in the DNA binding domain (DBD) influencing its thermal stability. It would be\nnecessary to show thermomorphogenesis phenotypes in ARF19 W330L and ARF19 R297W\nvariant. And, this explanation is based solely on predictive analysis and does not clarify how the\ncritical sites contribute to the protein's acquisition of thermal stability. Perhaps it affects nuclear\ntranslocation or downstream signal transduction. Is it the case that the protein undergoes\nstructural changes under heat, making it less susceptible to degradation?\nUnderstanding the structural basis for ARF thermal stability is outside the scope of this study,\nalthough we hope that the variants we identified in our screen may lend clues to future studies in\nthis area. This work also complements recent work identifying a potential degron in ARF2,\nwhich lies near the sites we identified.\nAs the protein level changes are the main molecular phenotype of this manuscript, all\nimmunoblots should be performed at least three times and quantification of the intensity\nshould be performed with error bars included.\nWe now provide quantification of signal from multiple replicates for each western blot. These\nare provided next to the representative immunoblot.\nThe immunoblot experiments in Fig. 1m should be repeated with proper loading control.\nWe appreciate the reviewer’s concern regarding equal loading across samples. Howeveer, we\nnote that inclusion of a conventional loading control is not feasible or appropriate for\ncycloheximide chase experiments due to the nature of the assay. CHX inhibits protein synthesis\nglobally, and thus the abundance of loading controls will change dynamically (and in\nuncharacterized ways) during the experiment.\nFig.2c-e were not referred in the manuscript.\nWe have now cited 2C-E in the manuscript.\nLine145, essential information is lacking to link to the statement “Our data is consistent\nwith the possibility that ARF7 and 19 display upper critical solution 145 temperature\n(UCST) behavior”.\nWe have expanded this section to make it more apparent how solubility and temperature related\nin UCST systems. Our text now reads: “Our data is consistent with the possibility that ARF7 and\n19 display upper critical solution temperature (UCST) behavior (Fig. 2a), often displayed by\nPrion-like domain (PrLD)-containing proteins 23-25. In UCST phase separation systems,\nmolecules such as ARF7 and 19 display increased solubility at elevated temperature and thus the\ndilute concentration of the molecule increases with increasing temperature (Fig. 2a). Enthalpy-\ndriven UCST behavior can be mediated by aromatic sidechain associations. Analysis of the\nARF7 and ARF19 intrinsically disordered regions reveals residues compatible with UCST\nbehavior (Figs. S5, S6). Indeed, Borniego et al. (accompanying manuscript) observe\ntemperature-regulated ARF7 and ARF19 solubility in root tissues and in yeast. In sum, our data\nare consistent with a model in which temperature regulates the levels of nuclear ARF7 and\nARF19 protein not only by altering protein stability but also by increasing protein solubility.”\nLine76-77 Fig.1d,e rather than Fig.1h, i should be referred to.\nThanks for catching this; it has been corrected.\nLine200-201, Fig.3f rather than Fig.3g should be referred to.\nThanks for catching this; it has been corrected.\nWe thank the reviewers for their constructive comments. We have made significant\nchanges that improve our manuscript. In particular, we have reorganized and rewritten our\nmanuscript to emphasize the molecular thermoresponsive behavior of ARF7/19. Further,\nwe have softened our interpretations of the physiological relevance of the molecular\nfeatures in the mutants in Col-0 and the interactions with other known pathways. Overall,\nwe feel that this stronger emphasis on the molecular phenotypes observed results in a\nstronger manuscript with well-supported claims."}, {"round": 2, "reviewer_comments": {"reviewer_1": "While I acknowledge the biochemical/molecular data presented in this manuscript, my overall assessment remains unchanged. The authors unsuccessfully attempt to connect a molecular phenotype with a thermo-inducible growth response. I do not see a convincing causal relationship between the observed molecular condensation/protein stability phenotypes and the claimed thermoresponsive growth behavior. In my view, the narrative is fundamentally flawed for several reasons:\na) I strongly doubt that arf7,19 mutants exhibit a genuine defect in thermoresponsive hypocotyl growth. Without clear evidence of such a phenotype, the described molecular condensation and protein stability features lack physiological relevance (see detailed explanation below).\nb) Conceptually, the authors’ interpretations make no sense. They fail to demonstrate a causal link between condensation behavior and growth phenotype; in fact, the data and the interpretations are often in contradiction. It even appears that the data directly undermine the manuscript’s main conclusions (see detailed explanation below).\nIn summary, readers are left with a thorough characterization of the temperature-sensitive molecular behavior of ARF7 and ARF19. However, despite the authors’ efforts to argue for a causal connection to thermoresponsive seedling growth, the conceptual framework is inconsistent and unsupported by the data. Paradoxically, the data seem to contradict the authors’ own conclusions, a point that the authors do not appear to recognize. The consequences of the condensation behavior on thermoresponsive growth are not only overstated, but also misinterpreted. Physiological relevance of these findings remains unproven.\nExplanation of a)\nFirst of all, I initially questioned the physiological relevance of the relative growth response data because of their minimal differences to the wild type. To better understand these, I asked to show the absolute growth data (which should in any case always be provided for full transparancy). After the authors provided the requested absolute hypocotyl growth data in this revised version of the manuscript (unfortunately not for the majority of all other growth assays), it became clear that arf7,19 double mutants do not exhibit a defect at high temperature; at 28°C, mutant hypocotyls are actually significantly longer than those of the wild type. Notably, the mutant hypocotyls are already elongated at control temperatures. Given that light responses typically act to counterbalance temperature-induced hypocotyl elongation, there are inherent limits to further promoting growth at high temperatures. When a mutant already displays increased hypocotyl length under control conditions (here 20°C), it is unlikely that this line can elongate much further in response to elevated temperatures. In other words, This baseline difference dampens their temperature responsiveness, as these mutants would need to elongate much more than wild type at 28°C to offset this initial disparity\nConsequently, relative growth responses may appear reduced, which could be misinterpreted as a defect in temperature responsiveness.\nMy concerns in this regard were further reinforced by the data presented in the new Figure 1e. In this figure, the authors quantified cell lengths in different hypocotyl zones and at various temperatures. They report that, in the most elongation-prone middle zone, cells in the arf7,19 double mutants ‘start off’ longer and do not show a robust increase in length in response to elevated temperature. The authors interpret this as evidence that arf7,19 double mutants are unable to respond properly to high temperature. I strongly disagree with this interpretation. The data actually show that, at elevated temperatures, cell lengths in this zone are statistically similar between the double mutants and wild type. This indicates that cells in the arf7,19 mutants can achieve the same maximum length as those in wild type. The observed difference is present at control temperature, pointing to a developmental defect rather than a defect in temperature response. Interpreting this as a temperature response defect confuses cause and effect. Of note and in contrast to Wilkinson et al., the authors of the accompanying manuscript (Borniego et al.) appear to share my concerns regarding the impact of aberrant growth at control temperature on responsiveness to elevated temperature. They write in their rebuttal 'We do understand that, as indicated by the reviewer, calculating relative growth [...] would conflate changes in the response to warmth with variations in growth capacity at 20°C, making these effects indistinguishable.'\nAs such, the observed phenotypes are not due to an impaired response to high temperatures, but rather stem from a developmental defect already present under control conditions. This scenario makes it extremely challenging—if not impossible—to interpret relative growth data in a meaningful way. As a side note, Okushima et al. (2005, Plant Cell) have already shown numerous developmental defects in various combinations of arf7,19 mutant alleles at control temperatures.\nExplanation of b)\nConceptually, the authors’ interpretations are inconsistent and fail to establish a causal link between the condensation behavior of ARFs and the observed growth phenotypes. According to their model, low temperature induces condensation of ARFs in the cytoplasm, leading to their inactivation by preventing nuclear localization and transcriptional regulation. If this were the case, one would expect wild type plants and ARF loss-of-function mutants to display similar phenotypes at low temperatures. However, the data show that arf7,19 double mutants have significantly longer hypocotyls than wild type at 20°C, directly contradicting this expectation.\nFurthermore, the literature reports numerous phenotypic defects for various arf7,19 allelic combinations even at control temperatures (e.g., Okushima et al., 2005), which should not occur if ARF7 and ARF19 were only active at elevated temperatures. The authors also argue that arf7,19 double mutants display a reduced temperature response based on relative growth data, implying that ARF7 and ARF19 are positive regulators of high temperature-induced hypocotyl elongation. Yet, the loss of these factors results in longer hypocotyls at both control and elevated temperatures. Hence, when functional ARF7 and ARF19 have a positive regulatory effect on hypocotyl elongation irrespective of temperature. How can this be the case when the condensation status of the proteins is so markedly different between the two temperatures?\nTaken together, the arf7,19 double mutants exhibit longer hypocotyls than wild type at both control (20°C) and elevated temperatures (28°C). I cannot imagine a clearer refutation of any proposed connection between the condensation status of these proteins, which differs profoundly between temperatures, and the observed growth response (longer at both temperatures). This fundamental contradiction undermines the main conclusions of the manuscript.\nAdditional comments:\n- Potential genetic redundance of additional class A ARFs with ARF7 and 19 that may mask a clear growth phenotype:\nThe authors were unable to provide growth phenotypes for additional class A ARF mutant lines. They argued that ARF5 is the class A ARF most closely related to ARF7 and 19. Are the authors sure that this is the case? It has been published several times that ARF5 diverges outside of the two pairs ARF7/19 and ARF6/8, which are more closely related to each other than to ARF5 (e.g., Okushima et al., 2005, Plant Cell; Remington et al., 2004, Plant Physiology). In any case, the authors only commented on arf5 mutants being seedling lethal. Why they did not perform the same hypocotyl growth assays for arf6 and arf8 mutants, both of them are perfectly viable (and most likely even more closely related to ARF7 and 19), was not mentioned (see also comment below to Fig 2k and i). What they did was to analyze molecular phenotypes of arf5 mutants. They argue that the similar condensation behavior observed in ARF5 supports functional redundancy among class A ARFs in thermomorphogenesis. However, this is not the case. While this observation suggests that thermoresponsive condensation may be a general feature of class A ARFs, it does not provide any evidence for a functional role in thermoresponsive growth. Condensation behavior could influence a variety of cellular processes, but as detailed above, there is no convincing evidence that it specifically affects thermoresponsive growth.\n- Lack of material description:\nUnless I missed it, the mutant arf7,19 alleles that form the center of this study have not been described at all in the Material and Methods section or anywhere else in the text. This is especially important because Okushima et al. (2005, Plant Cell) have reported several allelic combinations.\n- Insufficient decoupling of auxin biosynthesis and arf7,19 phenotypes\nThis concern was raised by two of the three reviewers. Reviewer 3 specifically suggested measuring auxin levels in the different genetic backgrounds, which would have been the most direct and effective way to address this issue. The authors, however, chose not to pursue this approach, justifying this with the inability to measure auxin levels in their current host laboratory. Given the corresponding author’s scientific background—Lucia Strader was trained in an auxin metabolism lab and is widely recognized as one of the most well-connected scientists in the auxin research community—this explanation seems rather unconvincing. Such measurements should have been feasible, at least through collaboration.\nInstead, the authors opted to use DII-Venus reporters. First, the DII-Venus expression has only been measured in wild type plants treated with biosynthesis inhibitors, but not in arf7,19 double mutants. Hence, the relevant genotype for this experiment is missing, rendering the results not really meaningful to begin with. Second, DII-Venus reporters do not directly measure auxin levels but rather report on auxin responses at the level of Aux/IAAs. As a result, these reporters can only provide an indirect indication of auxin levels, and their readout can be influenced by multiple factors downstream of auxin biosynthesis. Other available reporters, such as those fused to auxin biosynthesis genes or the direct auxin sensors (AuxSen by Herud-Sikimic et al., 2021, Nature - although I am not sure whether this reporter works outside roots), might have been a more appropriate choice if a direct measurement was not possible. In summary, the experimental approach chosen by the authors does not adequately address the concerns regarding the decoupling of auxin biosynthesis and arf7,19 phenotypes.\nl. 75-78: the authors write: 'However, when grown at 28°C, arf7 arf19 seedlings respond less robustly to elevated temperature than wild type in relative increase in hypocotyl elongation (Fig. 1b), displaying a mild reduction in final hypocotyl length (Fig. 1a,b) and petiole elongation (Fig. 1a,c), compared to wild type.' This statement is once again phrased in a misleading way. arf7,19 seedlings do not exhibit a mild reduction in final hypocotyl length. In fact, as clearly shown in Figure 1d with the absolute growth data, they display an increase in hypocotyl length at 28°C. Moreover, regarding petiole length, only relative data are presented. This raises the question: why do the authors consider transparency essential for hypocotyl length but not for petiole length? Without the corresponding absolute data, it is not possible to make any definitive claims about an increase or decrease in petiole length at 28°C.\nl. 78ff: See comment to new Fig. 1d above.\nFig. 1g: Interpretation of the data is challenging because only relative growth values are provided, with absolute data missing. Regarding the statistical analysis, while it is common practice to use a simple one-way ANOVA to compare relative data, this approach is not appropriate for the following reasons. First, it ignores the variance from the control treatment (in this case, the control temperature). Second, transforming warm temperature data into a percentage of control distorts the variance structure (and it is unclear whether the normalization was based on the mean or median of the 22°C hypocotyl length).\nThe proper approach would be to perform a two-way ANOVA on log-transformed data from the complete dataset (which should be provided, at least in the supplementary materials), and to report the interaction p-value for pairwise comparisons with appropriate correction for multiple testing. This method would account for the overall variance across all treatment and genotype levels.\nThis recommendation applies to all plots where relative data have been subjected to statistical comparison.\nl. 106-107: ' This increase in ARF7 and ARF19 protein accumulation aligns with their role in mediating the growth effects of elevated temperature'. I fully agree with all other statements in the paragraph. However, based on the arguments outlined above, I believe this important statement is not supported by the data.\nl. 122-124 + Fig. 2i: 'ARF7 and 19 are closely-related, with ARF5 as the next most-closely related to ARF7 and 19. ARF6 and 8 are additional Class-A ARFs in a separate clade from ARF7, 19, and 5.' See above, and please explain why the phylogenetic tree shown in Figure 2i depicts different relationships than those reported by Remington et al. (2004, Plant Physiology) and Okushima et al. (2005, Plant Cell). Both of these foundational studies performed phylogenetic analyses on ARFs and consistently identified ARF5 as the outlier in this clade, with ARF7 and ARF19 being more closely related to ARF6 and ARF8. Could you clarify the reasons for this discrepancy in your analysis?\nl. 130-133 + Fig. 2k,i: 'We did, however, find that arf6, arf7, and arf19 display reduced thermomorphogenesis in the form of reduced thermoresponsive hypocotyl growth rates (Fig. 2k), as did the arf6 arf7, arf7 arf8, and arf7 arf19 mutants (Fig. 2l). These data suggest that multiple Class-A ARFs contribute to thermomorphogenesis ...' --> I do not understand why the authors have not simply presented standard hypocotyl length data. For readers to properly assess whether other class A ARFs play a role in thermomorphogenesis, it is essential to see the same assays used for the target genes in this study. Instead, the authors present hypocotyl growth rates of 5-day-old seedlings that were pulsed with far-red light prior to a 10-hour dark incubation at either 20°C or 28°C. This approach essentially compares apples to oranges and raises concerns about the experimental design. It prompts the question of whether these lines failed to exhibit notable phenotypes in conventional hypocotyl growth assays, leading the authors to test a variety of alternative conditions until they found something—however unconventional—that could be interpreted as supportive of their claims. Such an unusual setup may affect general hypocotyl growth, but it is equally plausible that it does not reflect thermoresponsive hypocotyl elongation. As a result, the data provided do not allow readers to draw meaningful conclusions about general thermomorphogenic responses.\nFig. 3o: Why was no statistical testing applied here?\nFig. 4h: Please provide absolute growth data to allow a reasonable interpretation of the data. Are the ARF19W330L and R297W lines specific for temperature-responsive behavior or do they also display general (auxin-related) growth defects?", "reviewer_2": "The authors have provided additional data regarding the effects of high temperature on ARF protein accumulation and its independence of canonical auxin signalling.\nQuantifications provided for many blots with antibody against native ARF7 protein (Fig. 2d, 2p, 3q, S5b-d), which were not present in the original manuscript, show a large variation in protein increase at elevated temperature. For instance, in about half of all samples shown in Fig. 3o (wild type and mutants), no clear increase at 32 °C can be observed. Increased ARF accumulation appears more consistent with tagged overexpression lines (Fig. 2d, 2r), but even here, the variation is large in some cases (Fig. 2q). This draws into question the choice of some representative images (Fig. 2p, 3j-n, S5a). Importantly, it means that the (in)dependence of ARF accumulation from established temperature signalling pathways such as HY5 (Fig. 3j, S5c), ELF3/4 (Fig. 3l, S5d), phyB/PIF (Fig. 3m-o) and auxin (Fig. S5a, b) cannot be reliably assessed.\nI maintain that the manuscript holds a lot of valuable data. However, the high temperature-induced increase in ARF7/19 protein accumulation, and its independence from known temperature signalling pathways, are central conclusions of this study. Given the lack of robust protein data to support these conclusions, I cannot recommend acceptance of the manuscript in its current form.", "reviewer_3": "The authors have addressed all my concerns by performing additional experiments or providing explanations."}, "author_response": "We do not disagree with the reviewer that the arf7 arf19 mutant displays developmental defects at ambient temperature – this is to be expected for auxin signaling mutants, as they are required for all aspect of plant growth and development and indeed there have been numerous reports of these defects. Given this complexity, we have refocused our manuscript on the molecular consequences of temperature on ARF protein accumulation, which, unlike the subtle phenotypes of the arf7 arf19 mutant, are quite dramatic.\nFurther, as a part of the ongoing studies in our lab, we took advantage of the natural variation in ARF7 temperature-regulated accumulation to perform a genome-wide association study (GWAS) across a wider range of Arabidopsis accessions. Although this GWAS is not included in this manuscript, the results give us more confidence in the roles ARF7/19 thermoresponsive behavior is playing in thermomorphogenesis. From this natural variation screen, we identified new factors that 1) display natural variation in ARF7 thermoresponsive accumulation, 2) fail to demonstrate hyperaccumulation of ARF7 at elevated temperature, and 3) are strongly defective in thermomorphogenesis without generalized growth defects. Importantly, mutants defective in this identified gene display ARF7 accumulation like wild type at 22 C – thus, the defect is specifically limited to elevated ARF accumulation at elevated temperature. We are still actively working to determine the direct link between this factor and the ARF proteins and therefore have not included this work in the current study; however, we feel confident that this work will address many of the concerns of Reviewer #1 on the physiological relevance of the observed molecular phenotypes. This newly identified pathway does not appear to affect auxin responses at ambient temperatures, but solely the temperature-regulated accumulation of ARFs and the resultant thermomorphogenesis.\nNonetheless, we have now decreased our emphasis on the physiological relevance of the molecular effect of temperature on ARF accumulation, as we agree that assessing this with pleiotropic arf mutants is not sufficient to make strong claims.\nExplanation of b)\nIt has now been well-documented that ARF7 and ARF19 transcription factors are predominantly nuclear in young root meristems, and primarily in condensates in older cells that have ceased growth – this condensation attenuates ARF7 and ARF19 activity (see Powers et al, 2019 and the >250 papers that cite/follow up on this work). This is what occurs at 22C. There are likely many factors affecting ARF condensation, including posttranslational modifications, interactors, and environmental conditions, as has been well-described in the literature – there are many recent reviews on this topic.\nWe feel it is disingenuous of this reviewer to state that we claimed that ARFs are fully in condensates at 22C (we did not) and/or that we present a model to this effect, as we did not.\nFurthermore, the literature reports numerous phenotypic defects for various arf7,19 allelic combinations even at control temperatures (e.g., Okushima et al., 2005), which should not occur if ARF7 and ARF19 were only active at elevated temperatures. The authors also argue that arf7,19 double mutants display a reduced temperature response based on relative growth data, implying that ARF7 and ARF19 are positive regulators of high temperature-induced hypocotyl elongation. Yet, the loss of these factors results in longer hypocotyls at both control and elevated temperatures. Hence, when functional ARF7 and ARF19 have a positive regulatory effect on hypocotyl elongation irrespective of temperature. How can this be the case when the condensation status of the proteins is so markedly different between the two temperatures?\nTaken together, the arf7,19 double mutants exhibit longer hypocotyls than wild type at both control (20°C) and elevated temperatures (28°C). I cannot imagine a clearer refutation of any proposed connection between the condensation status of these proteins, which differs profoundly between temperatures, and the observed growth response (longer at both temperatures). This fundamental contradiction undermines the main conclusions of the manuscript.\nThank you for these comments. It seems that this reviewer is either not familiar with the tenets of protein phase separation or is willfully misunderstanding the body of literature that has accumulated on ARF condensation and regulation of ARF activity.\nProtein condensation can act to buffer protein concentrations (see Banani et al and related reviews). Because ARF7 and ARF19 form protein condensates, it is possible that temperature-regulated changes in accumulation might have no effect on the pool of active ARF protein (ie, nuclear / not in condensates). We do not make any claims about thermoresponsive ARF condensation and direct effects on growth; indeed, we did not perform any experiments to test this connection. Our purpose of examining the nuclear concentration of ARFs at elevated temperature was to determine whether elevated ARF accumulation at elevated temperature would also result in increased nuclear ARF or whether this system would be buffered. We have thoroughly explained this in the text.\nAdditional comments:\n- Potential genetic redundance of additional class A ARFs with ARF7 and 19 that may mask a clear growth phenotype:\nThe authors were unable to provide growth phenotypes for additional class A ARF mutant lines. They argued that ARF5 is the class A ARF most closely related to ARF7 and 19. Are the authors sure that this is the case? It has been published several times that ARF5 diverges outside of the two pairs ARF7/19 and ARF6/8, which are more closely related to each other than to ARF5 (e.g., Okushima et al., 2005, Plant Cell; Remington et al., 2004, Plant Physiology). In any case, the authors only commented on arf5 mutants being seedling lethal. Why they did not perform the same hypocotyl growth assays for arf6 and arf8 mutants, both of them are perfectly viable (and most likely even more closely related to ARF7 and 19), was not mentioned (see also comment below to Fig 2k and i).\nWe have removed the arf6 and arf8 mutant experiments from our manuscript, as there is some redundancy in the conclusions with those of the accompanying manuscript.\nHowever, we do expand with analysis of ARF6 and ARF8 protein accumulation, finding that all Class-A ARFs display thermoresponsive protein accumulation (Figure 1). In the manuscript, we state that the accumulation several A-ARF proteins is thermoresponsive. Further, we do not argue that ARF5 has a role in thermomorphogenesis, only suggest it is possible but not easily testable.\nWhat they did was to analyze molecular phenotypes of arf5 mutants. They argue that the similar condensation behavior observed in ARF5 supports functional redundancy among class A ARFs in thermomorphogenesis. However, this is not the case. While this observation suggests that thermoresponsive condensation may be a general feature of class A ARFs, it does not provide any evidence for a functional role in thermoresponsive growth. Condensation behavior could influence a variety of cellular processes, but as detailed above, there is no convincing evidence that it specifically affects thermoresponsive growth.\nWe did not demonstrate or make any claims about ARF5 thermoresponsive condensation, but rather that ARF5 protein accumulation varies with temperature.\nIn addition, we make no claims in the text about about thermoresponsive ARF condensation driving growth outcomes. Rather, we demonstrate that condensation is unlikely to buffer this system at elevated temperatures. We detailed text on this in the main text.\n- Lack of material description:\nUnless I missed it, the mutant arf7,19 alleles that form the center of this study have not been described at all in the Material and Methods section or anywhere else in the text. This is especially important because Okushima et al. (2005, Plant Cell) have reported several allelic combinations.\nWe have clarified in the figure legends that we characterize the arf7-1 arf19-1 mutant.\n- Insufficient decoupling of auxin biosynthesis and arf7,19 phenotypes\nThis concern was raised by two of the three reviewers. Reviewer 3 specifically suggested measuring auxin levels in the different genetic backgrounds, which would have been the most direct and effective way to address this issue. The authors, however, chose not to pursue this approach, justifying this with the inability to measure auxin levels in their current host laboratory. Given the corresponding author’s scientific background—Lucia Strader was trained in an auxin metabolism lab and is widely recognized as one of the most well-connected scientists in the auxin research community—this explanation seems rather unconvincing. Such measurements should have been feasible, at least through collaboration.\nIn our revised version of the manuscript, we de-emphasize this data as we more strongly focus on the molecular phenotypes (as suggested by the editor).\nFurther, while Lucia Strader did indeed train in the lab of Bonnie Bartel, the Bartel lab has never done any auxin analysis in-house (and rarely even by collaboration). Second, there are no fee-for-service facilities in the United States that can accurately measure auxin levels. Third, whereas Lucia Strader is indeed well-connected in the community, her conversations about addressing the reviewer concern of whether the auxin inhibitors ‘worked’ was widely viewed by experts in the community as a waste of time and resources (this would entail a week of work by a member of a collaborating lab), as the inhibitors used have been widely characterized for over a decade. Thus, these conversations with potential collaborators resulted in agreement that this was best addressed with a simple assay using DII-Venus.\nInstead, the authors opted to use DII-Venus reporters. First, the DII-Venus expression has only been measured in wild type plants treated with biosynthesis inhibitors, but not in arf7,19 double mutants. Hence, the relevant genotype for this experiment is missing, rendering the results not really meaningful to begin with. Second, DII-Venus reporters do not directly measure auxin levels but rather report on auxin responses at the level of Aux/IAAs. As a result, these reporters can only provide an indirect indication of auxin levels, and their readout can be influenced by multiple factors downstream of auxin biosynthesis. Other available reporters, such as those fused to auxin biosynthesis genes or the direct auxin sensors (AuxSen by Herud-Sikimic et al., 2021, Nature - although I am not sure whether this reporter works outside roots), might have been a more appropriate choice if a direct measurement was not possible. In summary, the experimental approach chosen by the authors does not adequately address the concerns regarding the decoupling of auxin biosynthesis and arf7,19 phenotypes.\nWe show that DII-Venus experiment as an indirect measurement of auxin biosynthesis at temperature to show that our inhibitors do in fact block auxin biosynthesis. Applicaton of these inhibitors alters hypocotyl elongation in wild type, in the YUC1 overexpression line, and in arf7 arf19, consistent with these auxin biosynthetic inhibitors altering auxin levels in all tested genotypes.\nUnfortunately using Auxsen is not ideal for this experiment, as it does not perform well in hypocotyls (per conversations with Xander Jones). In any case, we have de-emphasized this point in the manuscript, moving this data to supplemental.\nl. 75-78: the authors write: 'However, when grown at 28°C, arf7 arf19 seedlings respond less robustly to elevated temperature than wild type in relative increase in hypocotyl elongation (Fig. 1b), displaying a mild reduction in final hypocotyl length (Fig. 1a,b) and petiole elongation (Fig. 1a,c), compared to wild type.' This statement is once again phrased in a misleading way. arf7,19 seedlings do not exhibit a mild reduction in final hypocotyl length. In fact, as clearly shown in Figure 1d with the absolute growth data, they display an increase in hypocotyl length at 28°C. Moreover, regarding petiole length, only relative data are presented. This raises the question: why do the authors consider transparency essential for hypocotyl length but not for petiole length? Without the corresponding absolute data, it is not possible to make any definitive claims about an increase or decrease in petiole length at 28°C.\nWe have made this change to the text.\nAt the request of the editor, we have revised our manuscript to de-emphasize the growth phenotypes of arf7 arf19; this data has now been removed from the manuscript.\nl. 78ff: See comment to new Fig. 1d above.\nAt the request of the editor, we have revised our manuscript to de-emphasize the growth phenotypes of arf7 arf19; this data has now been removed from the manuscript.\nFig. 1g: Interpretation of the data is challenging because only relative growth values are provided, with absolute data missing. Regarding the statistical analysis, while it is common practice to use a simple one-way ANOVA to compare relative data, this approach is not appropriate for the following reasons. First, it ignores the variance from the control treatment (in this case, the control temperature). Second, transforming warm temperature data into a percentage of control distorts the variance structure (and it is unclear whether the normalization was based on the mean or median of the 22°C hypocotyl length).\nThe proper approach would be to perform a two-way ANOVA on log-transformed data from the complete dataset (which should be provided, at least in the supplementary materials), and to report the interaction p-value for pairwise comparisons with appropriate correction for multiple testing. This method would account for the overall variance across all treatment and genotype levels.\nThis recommendation applies to all plots where relative data have been subjected to statistical comparison.\nFor this data (now Supplemental Figure 8), we now include absolute and normalized hypocotyl data. We have log-transformed the hypocotyl length data and performed a three-way ANOVA to account for the variables of genotype, temperature, and treatment.\nl. 106-107: ' This increase in ARF7 and ARF19 protein accumulation aligns with their role in mediating the growth effects of elevated temperature'. I fully agree with all other statements in the paragraph. However, based on the arguments outlined above, I believe this important statement is not supported by the data.\nWe have softened this claim and rewritten our manuscript to reflect the emphasis on the molecular phenotype.\nl. 122-124 + Fig. 2i: 'ARF7 and 19 are closely-related, with ARF5 as the next most-closely related to ARF7 and 19. ARF6 and 8 are additional Class-A ARFs in a separate clade from ARF7, 19, and 5.' See above, and please explain why the phylogenetic tree shown in Figure 2i depicts different relationships than those reported by Remington et al. (2004, Plant Physiology) and Okushima et al. (2005, Plant Cell). Both of these foundational studies performed phylogenetic analyses on ARFs and consistently identified ARF5 as the outlier in this clade, with ARF7 and ARF19 being more closely related to ARF6 and ARF8. Could you clarify the reasons for this discrepancy in your analysis?\nDetailed phylogenetic analysis with a broad selection of sequenced plant genomes demonstrates that the ARF5/7/19 clade diverged from the ARF6/8 clade ~370 MYA, in the ferns (see image below from Mutte et al., 2018). The papers cited by the reviewer are from an earlier period before there was genomic support from multiple plant species. We have now cited this study in the manuscript to support statements about relationships amongst these proteins.\n[FIGURE REDACTED]\nl. 130-133 + Fig. 2k,i: 'We did, however, find that arf6, arf7, and arf19 display reduced thermomorphogenesis in the form of reduced thermoresponsive hypocotyl growth rates (Fig. 2k), as did the arf6 arf7, arf7 arf8, and arf7 arf19 mutants (Fig. 2l). These data suggest that multiple Class-A ARFs contribute to thermomorphogenesis ...' --> I do not understand why the authors have not simply presented standard hypocotyl length data. For readers to properly assess whether other class A ARFs play a role in thermomorphogenesis, it is essential to see the same assays used for the target genes in this study. Instead, the authors present hypocotyl growth rates of 5-day-old seedlings that were pulsed with far-red light prior to a 10-hour dark incubation at either 20°C or 28°C. This approach essentially compares apples to oranges and raises concerns about the experimental design. It prompts the question of whether these lines failed to exhibit notable phenotypes in conventional hypocotyl growth assays, leading the authors to test a variety of alternative conditions until they found something—however unconventional—that could be interpreted as supportive of their claims. Such an unusual setup may affect general hypocotyl growth, but it is equally plausible that it does not reflect thermoresponsive hypocotyl elongation. As a result, the data provided do not allow readers to draw meaningful conclusions about general thermomorphogenic responses.\nAt the request of the editor, we have have revised our manuscript to de-emphasize the growth phenotypes of arf7 arf19; this data has now been removed from the manuscript. For detailed morphometric analysis of arf mutants in response to elevated temperature, please see the accompanying manuscript.\nFig. 3o: Why was no statistical testing applied here?\nThis was an oversight on our part. In any case, further examination of the quantification of our blots led us to the decision to remove our quantifications. Without additional levels of controls, it is not possible to determine whether the western blot exposures are in the linear range (this would require a serial dilution of the protein being probed). Thus, we cannot make strong statements about the exact abundances of protein, but rather about the relative accumulation, using the loading controls provided.\nFig. 4h: Please provide absolute growth data to allow a reasonable interpretation of the data. Are the ARF19W330L and R297W lines specific for temperature-responsive behavior or do they also display general (auxin-related) growth defects?\nWe now provide the absolute growth data for the ARF19W330L and R297W lines (new Figure 3h), which partially rescue the arf7 arf19 developmental defects at 22C, but are defective in thermomorphogenesis.\nReviewer #2 (Remarks to the Author):\nThe authors have provided additional data regarding the effects of high temperature on ARF protein accumulation and its independence of canonical auxin signalling.\nQuantifications provided for many blots with antibody against native ARF7 protein (Fig. 2d, 2p, 3q, S5b-d), which were not present in the original manuscript, show a large variation in protein increase at elevated temperature. For instance, in about half of all samples shown in Fig. 3o (wild type and mutants), no clear increase at 32 °C can be observed. Increased ARF accumulation appears more consistent with tagged overexpression lines (Fig. 2d, 2r), but even here, the variation is large in some cases (Fig. 2q). This draws into question the choice of some representative images (Fig. 2p, 3j-n, S5a). Importantly, it means that the (in)dependence of ARF accumulation from established temperature signalling pathways such as HY5 (Fig. 3j, S5c), ELF3/4 (Fig. 3l, S5d), phyB/PIF (Fig. 3m-o) and auxin (Fig. S5a, b) cannot be reliably assessed.\nWe have softened the interpretation of our results. The data is indicative that ARF7 and ARF19s thermoresponsiveness is not reliant on these known pathways.\nIn addition, further examination of the quantification of our blots led us to the decision to remove our quantifications. Without additional levels of controls, it is not possible to determine whether the western blot exposures are in the linear range (this would require a serial dilution of the protein being probed). Thus, we cannot make strong statements about the exact abundances of protein, but rather about the relative accumulation, using the loading controls provided.\nI maintain that the manuscript holds a lot of valuable data. However, the high temperature-induced increase in ARF7/19 protein accumulation, and its independence from known temperature signalling pathways, are central conclusions of this study. Given the lack of robust protein data to support these conclusions, I cannot recommend acceptance of the manuscript in its current form.\nWe hope that our provided revisions have increased your enthusiasm for this work.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed all my concerns by performing additional experiments or providing explanations.\nThank you for your support."}, {"round": 3, "reviewer_comments": {"reviewer_1": "Let me briefly recap the development of this manuscript. It was originally submitted as a study proposing a new thermoresponsive mechanism controlling Arabidopsis thermomorphogenesis, a claim that, if robust, would clearly justify publication in a high-impact journal such as Nature Communications. After several rounds of review, the authors now appear to acknowledge that they substantially overstated the physiological effects of their mutants in earlier versions and that many of these effects are difficult to distinguish from general developmental defects. Consequently, they have removed a considerable amount of data and now explicitly restrict the scope of the work to cellular consequences of their mutant phenotypes (“Given this complexity, we have refocused our manuscript on the molecular consequences of temperature on ARF protein accumulation, which, unlike the subtle phenotypes of the arf7 arf19 mutant, are quite dramatic.”).\nIn this context, the strategy of introducing an unpublished GWAS study only in the rebuttal, to bolster their confidence in a physiological role of ARFs in thermomorphogenesis, is problematic. If these data are essential to support the central claim, they should be included, properly described, and peer-reviewed as part of the manuscript; if not, they should not be used to argue for physiological relevance in the response to reviewers.\nA further major concern in the previous versions of the manuscript has been the lack of a demonstrated causal link between ARF condensation behavior and thermomorphogenic growth responses. In their rebuttal, the authors suggest that this reviewer is either unaware of or deliberately misinterpreting the literature on ARF condensation (an unnecessarily personal and unprofessional tone, which detracts from constructive dialogue; similar to the comment about the corresponding author's conversations with colleagues about the waste of time of auxin measurements), and they state that they “do not make any claims about thermoresponsive ARF condensation and direct effects on growth” and again that “we make no claims in the text about thermoresponsive ARF condensation driving growth outcomes.” This is difficult to reconcile with the wording in the previous version of the manuscript, where they wrote that “we find that ARF7 and ARF19 promote thermomorphogenesis ... In particular, thermoresponsive ARF stability and condensation are rapidly altered by changing temperature.” It is hard to interpret this sentence as anything other than a suggestion that thermoresponsive ARF condensation promotes thermomorphogenesis, which they themselves defined as the “growth response to temperature” in the abstract.\nTaken together, the manuscript appears to have reached the review stage at Nature Communications partly on the basis of an overstated physiological significance that the authors now effectively downscale, offering instead a “refocus” on molecular phenotypes. This substantially lowers the impact of the work relative to the original framing. A more appropriate publication venue would likely be a specialized cell biology or molecular signaling journal, as the current version no longer delivers the major conceptual advance in understanding thermomorphogenesis that was initially implied.", "reviewer_2": "Wilkinson et al. have refocused their manuscript on molecular phenotypes of ARF7/19 temperature responses (protein accumulation and condensate formation), removing most phenotypic data. They also opted to remove all western blot quantifications from the manuscript, stating in their rebuttal that “it is not possible to determine whether the western blot exposures are in the linear range (this would require a serial dilution of the protein being probed). Thus, we cannot make strong statements about the exact abundances of protein, but rather about the relative accumulation, using the loading controls provided.”\nI agree that a dilution series would be required to confirm whether optical density measurements are in the linear range. However, unless the signal has become saturated (at which point neither the quantification nor the visual image can be trusted), an increase in protein levels between two samples on the same blot should still be detectable as an increase in optical density of the corresponding band (even if the increase is not proportional). Given the quantification shown in the previous version of the manuscript (which were all normalised to Col-0 at 22°C), a clear increase was detected in some samples (suggesting that saturation was not a problem here), but not others (see e.g. data points in Figures 2q, 3o, S5b-d). This still leaves me concerned about how representative the blots are that remain in the present version of the manuscript. If quantitative comparisons across different blots (and subsequent statistical analysis) are not feasible, the replicate blots and their individual quantifications should be shown in the supplementary material so the reader can gauge the variability in temperature-dependent protein accumulation for themselves. Any claims of statistical significance with regard to protein levels (line 256) should be removed."}, "author_response": "At the request of the editor, we indeed refocused our manuscript on the molecular phenotypes of temperature-responsive ARF protein accumulation. However, no data were removed from the previous revision, but rather a single figure moved to supplemental figures (again, at the request of the editor) to allow for a stronger focus on the molecular phenotypes. We stand behind all the data included in this work.\nWe do not feel that the GWAS is necessary to support the claims presented in this work. We included our unpublished GWAS in the rebuttal to partially assuage Reviewer #1’s concerns that there was no physiological relevance to temperature-responsive ARF accumulation, despite showing growth phenotypes demonstrating physiological effects. In particular, Figure 3h and 3i demonstrate that ARF19 variants that are resistant to the temperature effects on stability mostly rescue the morphological defects of arf7 arf19 but fail to rescue the arf7 arf19 morphological defects. We further show in Figure 6 that there is a correlation between ARF7 thermostability and thermomorphogenesis amongst natural accessions. This relationship is borne out in our (unpublished) GWAS.\nAgain, we maintain that temperature affects all five Class-A (activator) ARFs and thus we will be unable to visualize dramatic thermomorphogenic phenotypes without the ability to examine higher order mutants with arf5 (which are lethal). The mechanism we uncovered with our GWAS strongly regulates both thermomorphogenesis and the thermoresponsive stability of all Class-A ARFs. However, as this is a mechanism not previously implicated in temperature response, there is a great deal of work left to do to connect this mechanism directly to ARF control.\nWe have carefully read the manuscript and removed / softened any remaining language about altered condensation directly driving thermomorphogenesis.\nAs suggested, we have created supplemental figures with data for multiple replicates for each figure and have removed the statement of significance. Additional examples of each assay are now included in Supplemental figures 1, 2, 4, and 7. We would also like to note that our central claim of ARF temperature-responsive protein stability is supported by multiple assays, including immunoblot analysis (throughout the manuscript), time course microscopy (Fig 1d, 1e), and a protoplast-based stability assay (Fig 2a, 2b). There is uneven loading in Figure 1g, as seen in the actin lanes; we don’t believe this is temperature response of actin accumulation, based on the many westerns we have done with this protein. The differences in actin signal between 22 and 32 are quite slight compared to the dramatic differences in ARF signal between 22 and 32 in this figure. We feel that this data demonstrates temperature-responsive accumulation of these ARFs, even with the slightly uneven loading."}]

Decision letter