Thermosensory reconfiguration of the auxin transcriptional pathway to drive root cell growth

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

Abstract
Elevated temperatures enhance plant root growth. We find that cell elongation significantly contributes to this response. While mutations in the auxin transcriptional pathway impair warmth-induced cell elongation, exogenous auxin inhibits this growth. Intriguingly, warmth increases auxin levels and the nuclear accumulation of TIR1/AFB2/AFB3, alongside Aux/IAA stabilisation. This apparent paradox is explained by the concurrent increase in nuclear AFB1, which stabilises Aux/IAAs and promotes cell elongation. Notably, despite enhancing Aux/IAA stability, warmth also promotes ARF transcriptional activity. ARF7/19 are essential for warmth-induced cell growth and its inhibition by exogenous auxin. Warmth directly modulates ARF7/19 by reducing oligomerisation and cytoplasmic condensation, thereby enhancing their nuclear accumulation. This mechanism effectively repurposes the auxin pathway to regulate root cell growth in response to temperature.

Introduction
Temperature is a strong driver of evaporative demand because it increases vapour pressure deficit between the sub-stomatal cavity of plants and the atmosphere. Soil warming accelerates evaporation from the upper soil layers, reducing hydraulic conductivity and water accessibility to the plants. Thus, increasing temperatures enhance transpiration and reduce absorption, compromising the water status of plants. Elevating temperatures up to ~30°C has been shown to increase primary root growth in crops such as maize1 and sunflower2. This extended primary root length is a trait that can confer drought tolerance under specific conditions3.

In Arabidopsis thaliana, rising temperature increases the number of root cells by elevating the levels of auxin to stimulate its transcriptional pathway4,5. Warm temperatures also enhance root cell expansion4,6–9. It is unclear how the cell expansion response is compatible with the aforementioned elevation in auxin because high auxin inhibits root cell growth10. The effect of high auxin results from the activation of a rapid cytoplasmic branch initiated by the AUXIN SIGNALING F-BOX 1 (AFB1) receptor and a slower nuclear branch, mediated by TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AFB2/AFB3, which alkalinise the apoplast to inhibit cell elongation11–14. These pathways have preponderance over the rapid branch initiated extracellularly, which acidifies the apoplast and promotes growth15–17. In this study, we show that temperature hijacks the canonical auxin pathway, modulating the nucleocytoplasmic partitioning of AFB1, AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 to promote root cell growth.

Results
Warm temperature increases elongating cell number and cell length to promote root elongation
Root growth is a product of cell division, cell progression to rapid elongation, and the rate of cell elongation itself. In seedlings subjected to a 12-hour light/dark cycle for the shoot while the root remained in darkness without sucrose (two conditions that affect root growth18–20), increasing the temperature from 20°C to 28°C consistently stimulated primary root growth (Fig. 1a). This stimulation was due to both an increase in the number of cells (total number of root cells that contribute to root length, i.e. excluding meristem cells, Fig. 1b) and an increase in average cell length (calculated as the slope of root length plotted against cell number, Fig. 1c). Warmer temperatures decreased meristem size and cell number (Fig. 1d, e). After 12 hours at the warmer temperature, the elongation plus transition zone was longer despite the smaller meristem (Fig. 1f). By 24 hours, the length difference in the elongation and transition zone was no longer apparent, but the differentiation zone was longer at 28°C compared to 20°C (Fig. 1f, note the different scales for each zone). Furthermore, warm temperature increased the rate of cell division within the meristem (Fig. 1g). This, coupled with the reduced meristem size and cell number, and the transient increase of the transition plus elongation zone, indicates a more rapid passage from the meristematic to the elongation zone. Thus, elevated temperature enhances primary root elongation by affecting all three fundamental growth processes. An increase in cell number was responsible for 64% of the root elongation response during the first 72 hours of warm temperature exposure, with an increase in cell size accounting for the other 36% (see “Methods” for calculation details). The increase in cell number was further broken down: 49% of the total response was due to an accelerated transition of cells from the meristem to the elongation zone (13 of 17 cells, Fig. 1b and e), and 15% was due to cell division (the remaining 7 cells).

Warm temperature prolongs the elongation of differentiating cells
Since we observed differences in average cell length, we investigated the length of cells at different developmental stages. In dicotyledonous plants, root growth is limited by the external tissues21. The epidermal cells within the early differentiation zone (where the vasculature and the incipient root hairs first become visible, Supplementary Fig. 1) did not show changes in size with the temperature treatment (Fig. 1h). However, whilst more mature cells within the differentiation zone nearly ceased their elongation at 20°C, those shifted to 28°C continued growing (Fig. 1i), apparently elevating the threshold size at which primary root cells of Arabidopsis stop elongating22.

The promotion of root cell growth by warm temperature requires the nuclear pathway of auxin
The canonical, transcriptional pathway of auxin signalling involves hormone perception by the TIR1/AFB components of SCFE3 ubiquitin ligase complexes and their AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) co-receptors. Aux/IAAs become targeted to degradation relieving the inhibition imposed on ARFs to promote auxin-induced transcription of numerous gene targets, including SMALL AUXIN UP-REGULATED RNA (SAUR) genes23–26. Nuclear AFB1 cannot replace TIR1 function13 and inhibits canonical auxin signalling, presumably acting as a dominant-negative12. Recent observations indicate that in the presence of auxin, TIR1/AFB interacting with Aux/IAA have adenylate cyclase activity, and that the resulting cAMP is indispensable for auxin-induced transcription by ARFs27,28.

We measured the length of epidermal cells within the advanced differentiation zone in more than 50 mutants of genes involved in the canonical auxin pathway. Some mutants showed a cell-length phenotype at 20°C (Fig. 2a), and in all cases these genetic effects were already present in cells of the early differentiation zone (Fig. 2b); i.e., they did not occur in the zone of response to temperature. Furthermore, cell length at 20 °C did not correlate with their subsequent response to warm temperature (R2=0.05, not significant), indicating that it did not intrinsically constrain this response.

Affecting with selected mutations any of the steps of the auxin canonical pathway, i.e., auxin synthesis, TIR1/AFBs-Aux/IAA co-receptors, ARF transcription factors, or SAURs, reduced the response to temperature (Fig. 2a). None of the mutations enhanced the response to temperature. These results provide unequivocal evidence for the need of an intact auxin canonical pathway in the root cell-length response to temperature. Mutations affecting the plasma membrane H+-ATPases (AHA1/2), which work downstream multiple auxin pathways, or their positive TRANSMEMBRANE KINASE 1 (TMK1)/TMK4 regulators15,16 impaired the temperature response (Fig. 2a).

We found that mutations in afb1, arf7, and arf19 completely eliminated the cell-growth response to temperature (Fig. 2a), which contributes 36% to root growth promotion. However, the overall root growth phenotype of these mutants was not as severe as this value would suggest (Supplementary Fig. 2a, b). This finding highlights that root growth phenotypes are a result of the combined effects of the mutations on cell division, cell progression to rapid elongation, and cell elongation responses. The arf7 arf19 double mutant, for example, exhibited a stronger cell number response to temperature than the wild type (Supplementary Fig. 2c), partially compensating for the lack of cell elongation response. This enhanced cell number likely stems from the larger root meristem29 (Supplementary Fig. 2d).

Exogenous auxin does not promote root cell elongation
Since the cell growth promotion by temperature requires the components of the canonical pathway, we investigated whether the activation of this pathway by exogenous auxin (Picloram) mimicked the effect of warmth. Contrary to the effect of warm temperature, Picloram reduced cell length (Fig. 3a). Picloram also failed to reduce meristem length (Fig. 3b). However, Picloram did mimic the effect of warmth on cell division (Fig. 3c). These results do not support the interpretation that cell and meristem length responses to warm temperature derive exclusively from increased auxin, as in the case of cell division5. Cell growth inhibition by Picloram required ARF7/ARF19 (Fig. 3d), which are also necessary for the growth promotion by warm temperature (see Fig. 2a), suggesting the intriguing possibility that these auxin signalling components might not be acting in the same mode during the response to warm temperature.

Incoherent effects of temperature on the signalling state of the transcriptional pathway
Prompted by the results of the genetic and pharmacological analyses, we investigated the effects of temperature on auxin signalling status in epidermal cells within the advanced differentiation zone (i.e., the cells where elongation responds to temperature) at three points within the pathway. To focus the analysis of auxin levels on these cells, we used the AuxSen reporter30. Consistently with previous studies of free auxin at the root tip5, in epidermal differentiating cells 28°C increased auxin levels compared to 20°C (Fig. 4a, and Supplementary Fig. 3). The effect was also observed in the cortex/endodermis and vascular tissues (Fig. 4b). Warm temperature did not increase auxin in isolated root segments corresponding to the differentiation zone, suggesting that the origin of the additional active auxin was not local (Fig. 4c). Unexpectedly, warm temperature enhanced the fluorescence driven by DII-VENUS31, which indicates that warm temperature reduced auxin signalling at the level of Aux/IAA proteins (Fig. 4d). This response is opposite to that we observed with the same reporter in hypocotyl cells32 (Supplementary Fig. 4). Downstream of Aux/IAAs, warmth enhanced DR5:GFP fluorescence (Fig. 4e), indicating stronger activity of the ARF transcription factors33.

These results indicate that a temperature input reduces auxin signalling between hormone levels and Aux/IAAs, and an additional temperature input regains the strength of the signalling current downstream of Aux/IAAs. We reasoned that these temperature inputs could plausibly act on TIR1/AFBs and ARFs and tested these hypotheses in subsequent experiments.

Warm temperature increases AFB1 nuclear abundance
We speculated that warm temperature might reduce the abundance of TIR1/AFB2/3 accounting for the increased DII-VENUS (Fig. 4d). Contrary to the expectations, warmth increased the nuclear levels of fluorescence driven by pTIR1:TIR1, pAFB2:AFB2 and pAFB3:AFB3 transgenes fused to mCITRINE11 (Fig. 5a–c). The response of TIR1 is consistent with previous reports showing that warm temperatures stabilise TIR134.

The reduced promotion of cell elongation by warmth in afb1 (Fig. 2c) was initially surprising because AFB1 is predominantly present in the cytoplasm where it inhibits root growth11–13. However, warmth also increased the nuclear levels of fluorescence driven by pAFB1:AFB1 (Fig. 5d). A more detailed analysis revealed that AFB1 forms cytosolic condensates (Fig. 5e). Warmth only slightly increased AFB1 in the cytosol and cytosolic condensates, revealing a large increase in its nuclear partitioning (Fig. 5e).

Since AFB1 opposes the action of other TIR1/AFB nuclear receptors12, enhanced nuclear AFB1 could account for the increased DII-VENUS at 28°C compared to 20°C (Fig. 1d). Supporting this hypothesis, the nuclear fluorescence driven by AFB1-mCITRINE and DII-VENUS correlated across different temperatures (Fig. 5f), and across different tissues, because in contrast to mature differentiating cells, in meristematic cells, warmth increased neither AFB1-mCITRINE nuclear partitioning nor DII-VENUS (Supplementary Fig. 5). More importantly, the promotion of the DII-VENUS signal by warm temperature was absent in the afb1 mutant background, providing genetic evidence for the model where the nuclear shift of AFB1 protects Aux/IAA under warm conditions (Fig. 5g). Consistently with the reduction in the DII-VENUS signal, indicative of lower abundance of the repressors, the afb1 mutant showed enhanced fluorescence driven by DR5:GFP (Fig. 5h). However, in contrast to DII-VENUS, DR5:GFP did respond to temperature consistently with the idea that this environmental cue inputs downstream Aux/IAAs.

Complementation of the afb1 mutant with AFB1 fused to either a nuclear exclusion signal (NES)12 or a nuclear localisation signal (NLS)12 abolished the temperature response (Fig. 5i). These observations support the requirement of nuclear AFB1 and suggest that excessive nuclear accumulation might disrupt the balance by promoting the over-accumulation of Aux/IAAs and/or inhibiting cAMP synthesis by TIR1/AFBs, among other potential mechanisms. The dynamic, temperature-induced increase in AFB1 nuclear abundance is essential for the cell-growth response.

Warmth rapidly and reversibly increases nucleus/cytoplasmic condensate partitioning of ARF7/19
The inconsistent responses of DII-VENUS and DR5:GFP suggest that temperature influences the system downstream of Aux/IAAs. Consequently, we investigated the subcellular dynamics of ARF7/19, transcription factors essential for warm temperature-induced cell elongation (Fig. 2a). Both ARF7-VENUS and ARF19-VENUS undergo phase separation to form inactive cytoplasmic condensates35,36. In epidermal cells within the advanced differentiation zone, exposure to warmth led to a sustained increase in nuclear ARF7-VENUS and ARF19-VENUS (Fig. 6a, b). This increase was attributed to three changes with distinct kinetics: first, their release from cytoplasmic condensates (Fig. 6a, b, occurring at 24h for ARF7-VENUS and 4h for ARF19-VENUS); second, an increase in their overall cellular levels (Fig. 6a, b, observed at 48h for ARF7-VENUS and 24h for ARF19-VENUS); and third, their enhanced partitioning into the nucleus (particularly notable for ARF7-VENUS at 4h, before any apparent changes in condensate or cellular levels). While the accompanying paper by Wilkinson et al. explores the mechanisms regulating ARF7/19 protein stability, here we focus on their ARF7/19 release from the cytoplasmic condensates. Warmth induced a rapid shift in the nucleus/cytoplasmic condensate partitioning, a process that was reversible upon returning the seedlings to a mild temperature (Fig. 6c-d).

The nuclear fluorescence driven by the ARF7/19-VENUS transgenes increased, the fluorescence of the cytoplasmic condensates decreased, and their ratio increased between 9 and 34°C (Fig. 6e, f), indicating that the subcellular distribution of ARF7/19 conveys information over a wide range of temperatures. Consistently with this observation, the arf7 and arf19 mutants showed reduced cell length at 34°C (Fig. 6g), i.e., even at a temperature that reduced cell elongation by a different mechanism.

Warm temperature also increased nuclear ARF7/19-VENUS fluorescence in the cells of the elongation zone (Fig. 6h) but not in the cells of the meristematic zone (Fig. 6i), suggesting that a differential sub-cellular dynamic could be involved in ARF7/19 modulated transition between these two zones (see Supplementary Fig. 2c).

Warm temperature reduces ARF7/19 oligomerisation
Oligomerisation of ARF7/19 drives the formation of multivalent higher-order structures that promote cytoplasmic condensate formation, a process facilitated by an intrinsically disordered region35–37. Mutation of a conserved lysine residue (K962) within the ARF7/19 C-terminal type I/II Phox and Bem1p (PB1) domain impairs this oligomerisation both in vitro and in vivo35,38,39. Consequently, YFP-ARF19K962A shows severely reduced (but does not fully prevent) cytoplasmic condensate formation35,36. Given that the in vitro diffusion interaction parameter of ARF7 PB1 domains increases ~10-fold between 10°C and 40°C40, we hypothesised that a temperature-induced reduction in oligomerisation could be a primary driver of cytoplasmic condensate disassembly (Fig. 7a). Consistent with this, warm temperatures did not disassemble cytoplasmic condensates of the oligomerisation-deficient YFP-ARF19K962A, despite the fact that the condensates were easily detectable in the mutant line (Fig. 7b).

ARF7/19-EGFP formed discrete foci when expressed in yeast (Fig. 7c). These foci showed partial recovery after photo-bleaching with a kinetics that resembles that observed in plant cells35 (Fig. 7d). Increasing temperature above 20°C progressively dissolved the condensates (Fig. 7c, d). In plant cells, an E3 ubiquitin ligase36 and proteins acting as molecular chaperones41 facilitate the disassembly of ARF7/19 condensates, therefore, the observation in yeast is important because it indicates that warmth-induced disassembly of ARF7/19 condensates does not require any other plant protein.

The degree of ARF19 condensate multimerisation ranges from >10-mers at the core to dimers and monomers at the edge. To investigate ARF7/19 oligomerisation out of the condensates, we employed photon-counting histogram analysis to assess molecular brightness42,43. Cytoplasmic ARF7-VENUS displayed higher brightness at 22°C, which decreased at 34°C (Fig. 7g). This demonstrates that temperature can influence oligomerisation not only in vitro but also within the differentiating root-cell environment. Specifically, warmer temperatures reduced the molecular brightness of cytoplasmic ARF7-VENUS, but not in the nucleus, where ARF7 is predominantly monomeric35 (Fig. 7g, Supplementary Fig. 6). Given that genetically impairing oligomerisation facilitates nuclear accumulation35, this shift towards a monomeric state in the cytoplasm might facilitate its nuclear migration (Fig. 7a). This suggests a plausible mechanism for the observed increase in nuclear ARF7 during the early response (4h) to warmth, independent of significant changes in overall cellular abundance or detectable cytoplasmic condensate disassembly. Outside of the condensates in the cytoplasm, YFP-ARF19 and the non-oligomerising mutant YFP-ARF19K962A exhibited similar brightness, indicating that ARF19 exists predominantly as a monomer and therefore cannot respond to temperature after dissociating from the multimeric condensates (Supplementary Fig. 7).

Impaired promotion of cell elongation by warm temperature in seedlings expressing YFP-ARF19K962A
Seedlings expressing the mutant protein YFP-ARF19K962A exhibited impaired cell elongation in warm temperatures (Fig. 8a), suggesting that the mutant protein interfered with the response mediated by the wild-type gene. Interestingly, these same seedlings showed inhibited root cell growth when exposed to exogenous auxin (Fig. 8b), indicating that the mechanisms for these two treatments may be distinct. The shorter cell length observed in the control group at 20°C could be attributed to the larger nuclear concentration of YFP-ARF19K962A combined with endogenous auxin levels.

Methods
Plant material
We used seedlings of Arabidopsis thaliana accession Col-0 and the mutants and transgenic lines listed in Supplementary Data 1. We crossed the transgenic seedlings expressing the pDR5:NLS-3xGFP or p35S:DII-VENUS transgenes to the afb1-3 mutant and selected seedlings homozygous for these reporters (by confirming the lack of segregation in the F3 generation) and either for the wild-type AFB1 allele or the afb1-3 mutant allele (by using appropriate primers in the F2 and F3 generations). The primers used for genotyping are listed in Supplementary Data 2.

Growth conditions
Seeds were surface sterilised (3-h chlorine gas) and sown under sterile conditions on 7cm tall, 15cm width, 1.3cm depth clear boxes containing a 5cm tall, 15cm width, 0.8cm depth layer of 0.8% agar Hoagland solution, pH 5.8, topped with 2mm 0.8% agar, 0.6% activated charcoal powder (Supplementary Fig. 9a). The substrate and the walls of the box were covered with a black plastic. The boxes were incubated in darkness at 4°C for 4 days to reduce seed dormancy and obtain homogeneous germination. After the seed stratification step, the boxes with seedlings were transferred to a room at 20°C providing 12h white light (120 µmol m-2 s-1 between 400 and 700nm) / and 12h darkness for 5 days. Whilst the shoot was exposed to light the roots remained in darkness. One hour after the beginning of the photoperiod of day 6, the boxes were transferred to the indicated temperatures or remained as controls at 20°C, without changing the light conditions (time = 0). For Picloram and AuxSen experiments, the seeds were sown on black agar placed on top of black 96-well plates. After seed stratification, the seedlings were grown with their roots in darkness in contact with liquid Hoagland solution at 20°C under 12h photoperiod (Supplementary Fig. 9b). This procedure ensured better contact of the roots with the synthetic hormone, or the dexamethasone used to induce the expression of the AuxSen reporter.

Primary root length increment
Primary root length increments were calculated as the difference in length between 24, 48 or 72 h after the beginning of the temperature treatments and time = 0h. Immediately prior to the beginning of the temperature treatments, and at the indicated times, the boxes were briefly transferred to a room under dim green light, where the front black plastic cover of the boxes was removed to photograph the root area. The length of the primary root was measured using Fiji image software47.

Root cell length and cell number
The roots were fixed in 1% paraformaldehyde in PBS, mounted immediately on glass slides and visualised with the Axiostar Plus microscope (Carl Zeiss) using an A-plan 20x/0.45 objective. Digital images were acquired with a PowerShot G9 (Canon) camera. At least 20 cells were measured per seedling using Fiji image software47 and the average per root was used in the statistical analysis. In selected experiments, we quantified the number of elongating and elongated cells from the meristem to the root base. We then plotted total root length against this cell count. While root length measurements include the meristem, its contribution is negligible (<300 μm in 15-20mm roots). To quantitatively assess the contribution of cell number and cell size to root elongation, we performed the following calculations: The contribution of cell number was calculated by multiplying the cell number increment by the cell size at 20°C. The contribution of cell elongation was calculated by multiplying the cell-length increment by the number of cells at 28°C. The sum of these two contributions fully accounted for the temperature promotion of root elongation.

Meristem cell number
The number of root meristem cells was counted in confocal images of roots stained with calcofluor-white as the number of cortex cells in a file extending from the quiescent centre to the cell that was twice the length of the immediately preceding cell29,48.

Mitotic index
To determine the mitotic index, we used DAPI staining on fixed plants. We acquired five longitudinal optical slices of the meristem in a z-stack. For the analysis we calculated the of number mitotic cells relative to the total cell number for every layer of the z-stack in a region of interest (ROI) that was consistent across all samples. The mitotic index for each plant was determined by averaging the values from the five optical slices.

Picloram assay
Six-day-old seedlings were transferred to new plates containing either 350µL per well of liquid Hoagland solution (control) or 350µL of liquid Hoagland solution supplemented with 0.1, 0.5, 1, or 5µM Picloram. Finally, after 24 hours of treatment, the seedlings were harvested and fixed in 1% paraformaldehyde.

Plasmid construction and yeast transformation for heterologous expression
To generate constructs for heterologous expression in yeast cells, the coding sequences (CDS) of ARF7 and ARF19, lacking the stop codon, were amplified by PCR using Pfu DNA polymerase (PB-L Productos BioLógicos) and the attB1 ARF7c_FW/attB2 ARF7c_RV or attB1 ARF19c_FW/attB2 ARF19c_RV primer pairs (Supplementary Data 2). The resulting PCR products were then recombined into the Gateway™ pDONR™/Zeo vector (Life Technologies) using Gateway® BP Clonase™ II Enzyme Mix (Invitrogen). Subsequently, these entry clones were recombined with the yeast expression vector pAG414GPD-ccdB-EGFP49 to create C-terminal fusions with EGFP, using the LR Clonase II Enzyme Mix Kit (Invitrogen). The accuracy of the cloning was confirmed by DNA sequencing.

Verified recombinant plasmids were introduced into the Saccharomyces cerevisiae strain HH3 (genotype MATα, lys2, ade2, his3, trp1, ura3, leu2) via electroporation. Briefly, yeast cells were cultured to an optical density at 600nm (OD600) of 0.4–0.6 and harvested by centrifugation at 4000 × g for 10 min at 4°C. The cells were washed twice with Milli-Q water, followed by two washes with 10% (v/v) glycerol, and finally resuspended in 10% (v/v) glycerol. Then, 500µg of the appropriate plasmid was added to the resuspended cells, and electroporation was performed at 2.5kV, 25 µF, and 200 Ω. Following electroporation, cells were incubated in 1 ml of YPDA medium for 1 h at 28°C and then plated on selective synthetic complete medium supplemented with 1.39g/L Yeast Synthetic Drop-out Medium Y2001 (Sigma-Aldrich), 76mg/L histidine, 380mg/L leucine, and 76mg/L uracil. Plates were incubated at 30°C for 2–3 d, after which individual colonies were streaked onto fresh selective medium plates and incubated at 30°C until isolated colonies appeared.

Saccharomyces cerevisiae cells expressing EGFP-tagged ARF7 or ARF19 were grown overnight in liquid selective synthetic complete medium at 28°C, subjected to different temperature treatments (4°C, 20°C, 28°C, or 37 °C) for 40 minutes and fixed with 4% paraformaldehyde in PBS. The fixed cells were subsequently used for imaging analyses.

Confocal microscopy with GFP, mVENUS and mCITRINE reporters, DAPI and Calcofluor
We used Carl Zeiss confocal microscopes LSM 510Meta (8 channel spectral detector) and LSM 710/880 (both equipped with Quasar detectors). The GFP reporters were excited with a 488 nm laser and the emission detected from 500 to 530 nm. The mVENUS reporters were excited with 514 or 488 nm lasers and the emission detected from 525 to 560. The mCITRINE reporters were also excited with 514 or 488 nm lasers and the emission detected from 520 to 600 nm. DAPI and Calcofluor were excited with 405 nm laser and the emission detected from 420 to 510 nm for DAPI (e.g., Supplementary Fig. 10) and 414 to 440 nm for Calcofluor. For the analysis of nuclear and total cell fluorescence, images of the root were taken with Plan-Apochromat 20x/0.8 or W Plan-Apochromat 20x/1.0 objectives (Carl Zeiss). For the analysis of sub-cellular distribution of fluorescence confocal images of individual nuclei were taken with an EC Plan-Neofluar 40x/1.30 Oil objective (Carl Zeiss). We obtained images from three-four nuclei per root and averaged the information per seedling.

Image analysis was conducted by using the ImageJ47 software. Nuclei and condensates were segmented from duplications of each original image that were equally processed across each experiment using the same parameters (including background subtraction, smoothing, use of “watershed” for separating touching objects) to optimise the selection of Regions of Interest (ROIs) with the function “analyse particles”. The accuracy of the selection of ROIs was verified in every experiment and performed manually if necessary. The obtained ROIs were used to measure the original raw images. The same threshold selected for the application of the function “analyse particles” was used for the presentation of representative yeast cell images. We obtained similar results with either fresh material (taken from the treatment conditions immediately prior to the observations) or samples vacuum infiltrated in paraformaldehyde 1% in PBS at the same temperature used during the treatments50. The times indicated in the figures showing detailed kinetics (Fig. 5c, f) correspond to x min under treatment plus 5min during the infiltration, to account for the potential follow-up of the response before completion of the fixation process.

For yeasts we used a C Plan-Apochromat 63X/1.4 Oil DIC M27 objective (Carl Zeiss) and followed the same approach described for the analysis of images taken from roots. To avoid cell movements during the image acquisition, slides were treated with Poly-L-lysine. Except for FRAP experiments, yeast cells were fixed in paraformaldehyde 4% in PBS at the same temperature used during the treatments for at least 15 minutes and washed twice with 50mM ammonium chloride in PBS and resuspended in PBS.

Fluorescence recovery after photobleaching (FRAP)
For FRAP experiments we used live yeast cells at room temperature that were immobilised to the slides with Poly-L-lysine immediately before observation. Whole condensates were bleached with 488 nm Argon laser after 12 cycles of acquisition to determine the base-line pre-bleach fluorescence, and later every 0.234 seconds for 25 seconds to determine recovery. Images were acquired in 396×396 pixels and 0.09µm x 0.09µm pixel size, 16-bit and 0.63µs pixel dwell. For the analysis we determined three circular ROIs (diameter of 1.27µm). The first for the bleached condensate, the second for a reference condensate, and the third to analyse the background. Background fluorescence was subtracted from bleached and reference ROIs, and background-subtracted intensity was then divided by the background-subtracted intensity of the reference ROI to obtain relative fluorescence. Normalised intensity was calculated as (I(t) - I(t0)) / (I pre-bleach - I(t0)) where I(t) is the relative fluorescence of each time point, and I(t0) is the relative fluorescence immediately after bleaching.

FRET analysis of the AuxSen reporter
We used a LSM 510 Meta confocal microscope equipped with an eight-channel spectral detector and an EC Plan-Neofluar 40x/1.30 Oil objective (Carl Zeiss). Aquamarine was excited with a 405 nm laser and emission was detected simultaneously in 5 channels at 422-454, 454-486, 486-518, 518-550 and 550–583 nm of the spectral detector. The images were analysed with ImageJ47. To analyse FRET, we calculated for each nucleus the ratio between the mean fluorescence in the 518-550nm and the 454-486nm channels. We analysed three-four nuclei per root and averaged the information per seedling. For visual representations of FRET ratios we used the ImageJ plugin RatioloJ 4.051 to generate ratio-metric images from the same channels used for calculation. Using an infrared thermometer we did not observed differences in sample temperatures at the time of starting FRET measurements, indicating that the time taken to prepare the samples (~5min) was enough to equilibrate the differences in temperature caused by the treatments.

Fluorescence fluctuation spectroscopy
To compare the degree of oligomerisation in samples exposed to different temperatures we calculated the apparent molecular brightness plotting frequency against photon count per bin (photon counting histogram) and obtaining the variance and average intensity of the signal42,43. Temperature or sub-cellular localisation do not distort normalised molecular brightness values52; we therefore normalised the data to the values obtained for the control temperature. For the measurements, we used a LSM 880 confocal microscope with a C-Apochromat 40x/1.2W Korr FCS objective, the 488nm laser, the pinhole set to 1 airy unit (39μm) and the BIG2 GaAsP detector with 500–550 nm band-pass emission filter. The control samples were measured at 22°C, whereas the high-temperature condition was measured after equilibrating the incubation chamber and the plate to 34°C. Data were acquired by spot scanning in photon counting mode with an interval time of either 39µs (Fig. 7g, and Supplementary Fig. 7) or 1ms (Supplementary Fig. 6). The longer time was used because in the cytoplasm the number of molecules was relatively small. Scans without significant bleaching or instabilities were used for analysis. For this purpose, data were acquired during 12s and the first 2s were eliminated. We fitted these data to a Gauss curve by using GraphPad software (R2≥0.998) to estimate the variance and average intensity of the signal.

Statistics
We used liner regression tools of the GraphPad Prism software to compare the slopes of the roots exposed to 20°C and the roots exposed to 28°C in the experiments analysing the time course of root growth or cell growth in the wild-type Columbia (Fig. 1a, h, i). When the differences are statistically significant (F tests), at least the highest slope is statistically significantly different from zero. We used the same tool to analyse the relationship between the lengths of cells of different root zones across different genotypes (Fig. 2b), between confocal readings based on different reporters (Fig. 5f), or between confocal data and temperatures (Fig. 6e, f). We used the non-linear regression tool of the GraphPad Prism software to fit non-linear response curves (Fig. 3a, c).

We used the Student’s t tests (two tails, unpaired data) tool of the GraphPad Prism software for all comparisons between two means. In the confocal experiments comparing the effect of 28°C versus 20°C at one or more time points, the fluorescence data or fluorescence ratios were normalised to 1 by dividing the values by the average corresponding to 20°C in the same experiment. Since data are pooled from different experiments, this procedure reduced the small variability observed among different experiments but, more importantly, it ensures that the reader does not interpret variations in the 20°C as true temporal trends because the various time point data were not necessarily obtained the same day.

To analyse the interaction between genotype and temperature (Figs. 3d, 5g–i, 6g, and 8a) we used the multiple linear regression tool (stepwise model) of InfoStat (www.infostat.com.ar). The explanatory variables were temperature (20°C = 0, 28°C = 1), gene effect (wild type allele = 1, mutant allele = 0) and interaction (wild type allele at 28°C = 1, all the other combinations = 0). We show the significance of the interaction (t tests, two tails, unpaired data) and the differences between selected means according to Bonferroni tests (GraphPad Prism software). A comparable procedure was used to analyse the interaction between genotype and exogenous auxin treatment (Fig. 8b).

In Fig. 2a and Supplementary Fig. 2a–c, we present the response to temperature (28°C versus 20°C) of cell length, number of cells, or number of meristematic cells to facilitate the specific analysis of the response. For cell length and cell number, we show the absolute response between both temperatures. For meristem cell number, in addition to absolute responses, we used log-transformed data to obtain a relative response because the number of cells available in the meristem at 20°C affects the number of cells that can effectively transition to the elongation zone at 28°C. To calculate these responses we used the multiple linear regression tool of InfoStat (www.infostat.com.ar), incorporating one mutant at the time and the wild type data corresponding to the experiments that included this mutant. The explanatory variables were temperature (20°C = 0, 28°C = 1), gene effect (wild type allele = 1, mutant allele = 0) and interaction (wild type allele at 28°C = 1, all the other combinations = 0). A significant coefficient of gene effect indicates that the mutant has a significant effect at 20°C. A significant coefficient of interaction indicates that the mutant responds differentially to temperature when compared to Columbia. The t tests involved in each multiple regression analysis takes into consideration that different variables are tested simultaneously. When we show all the genotypes together in these figures, the significance values are not corrected for multiple comparisons because our goal was to test the null hypothesis of no involvement of the canonical pathway, and all the mutant phenotypes contribute to test this hypothesis. However, in Supplementary Data 3, we provide the p values corrected for multiple comparisons by using Benjamini–Hochberg procedure. Any statement of the article referring to selected mutants is based on the corrected p values.

The degrees of freedom were directly calculated by the software tools indicated for each test on the basis of the replicates indicated in the figures.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Overall evaluation\nBelen Borniego et al. present a detailed analysis of how the auxin co-receptor AFB1 and the two ARFs, ARF7 and ARF19, respond to temperature changes in Arabidopsis seedling roots. They describe interesting cellular behaviors suggesting thermosensory roles for ARF7 and ARF19 and a repressive effect of AFB1 on nuclear TIR1 signaling. In fact, they state that their ‚study identifies ARF7 and ARF19 as bona fide temperature sensors essential for root growth‘. I think this central claim of the paper is overstated for several reasons:\n1. It remains unclear if these cellular phenotypes—intriguing as they are—significantly impact root growth in response to temperature (these essential data are not provided). If ARF7 and ARF19 sense temperature without markedly affecting root elongation, this may limit the study’s contribution to understanding temperature-induced root growth.\n2. The conclusions rest exclusively on cellular data; incorporating additional methodologies would strengthen the proposed model.\n3. Compared to previously identified thermosensors, Belen Borniego et al. do not a) identify the direct targets of ARF7 and ARF19 involved in processing thermosensory information or b) establish causality between the thermosensory function and the described phenotypes. As it stands, it is unclear whether the arf7/19 mutant phenotypes are due to canonical nuclear auxin signaling triggered by an unknown thermosensor or whether the condensation and relocalization of ARF7/19 can bypass this signaling pathway to cause the observed phenotypes.\nFurthermore, Recent studies have highlighted temperature and auxin effects on root cell division, raising questions about how cell division and elongation contribute to temperature-responsive root growth—an aspect not addressed in this paper.\nOverall, while the study provides promising insights into AFB1, ARF7, and ARF19 behavior in response to temperature, its broader biological relevance remains uncertain.\nConceptual issues\nRoot Growth Relevance: As noted, it is unclear whether the observed cellular thermosensory responses in ARF7 and ARF19 significantly impact root growth. This is difficult to assess because root elongation data for the relevant mutants are not clearly presented (except in a rather obscured fashion in Fig. 1D: root length incremented plotted against differentiating cell length?; or 1E: length of differentiating cells?). The authors may be reporting detailed cellular responses to temperature in auxin-related proteins that, while molecularly interesting, might be transiently observable in a specific root zone but do not influence overall root growth, which would significantly weaken the impact of this study.\nAlignment with Previous Studies: The study’s findings appear somewhat at odds with prior work by Ai et al. (2023, EMBO J), who found that temperature did not significantly alter mature cell length in Arabidopsis roots but led to increased cell numbers. Belen Borniego et al. report that high temperatures prolong the elongation phase of differentiating cells, but they do not consider cell division effects. If differences in cell length during differentiation are transient and compensated for in mature cells, then the molecular phenotypes reported here may have limited impact on root growth. Without data on mature cell length, it’s challenging to judge if these observations reflect minor effects or if methodological differences might lead to inconsistent outcomes across labs.\nModel Support: The model of ARF7 and ARF19 as root thermosensors relies on subcellular localization observed via microscopy. These data are suggestive but would be more compelling if validated with an independent method, such as Western blotting on nuclear and cytoplasmic fractions from temperature-exposed samples, to substantiate the observed re-localization.\nFurthermore, and to expand on point 3 raised above, I am not convinced that the potential thermosensory function of ARF7 and ARF19 is causative for the observed cell length phenotypes. Have the authors considered the possibility that this could represent correlation rather than causation?\nFor instance, two well-established thermosensors, phyB and ELF3, demonstrate the following: a) they show dramatic phenotypes in temperature-responsive root growth when knocked out; b) the effect of thermosensing on their direct targets (such as PIF4 and the PIF4 promoter) is known; and c) their thermosensory function has been confirmed as causal. For example, the YHB line of phyB maintains phyB in a constitutively active conformation in the nucleus by preventing conformational changes. Similarly, the Q-repeats in ELF3 are critical for protein aggregation; mutant lines in both thermosensors clearly demonstrate that disrupting their thermosensory functions—whether through phyB conformational changes or ELF3 phase separation—leads to the expected phenotypes.\nIn contrast, there is no comparable evidence for ARF7 and ARF19 in this study. To establish a bona fide thermosensory function in root growth responses, it would be essential to have lines that a) disrupt the transition of ARF7 and ARF19 from the cytoplasm to the nucleus, or keep them constitutively localized in either compartment; or preferably, c) lines that regulate cytoplasmic condensate formation in a way that can be causally linked to the observed phenotypes. Finally, it must be established that the phenotypes associated with arf7 and arf19 mutants are not due to a disruption in canonical nuclear auxin signaling, which has been shown by multiple research groups to be crucial for root growth responses to temperature and is influenced by another, yet unidentified, thermosensor.\nSpecific comments\nAbstract\nThe abstract includes several statements that lack sufficient support:\n• The first sentence implies that root growth at elevated temperatures enables deeper rooting to access moist soil, which may not apply broadly. While this may be true for some species, others increase root surface in the topsoil to maximize moisture capture, which may be more relevant for Arabidopsis, whose roots rarely grow deeper than 5 cm in the field. Thus, this generalization might be inappropriate for the species in question.\n• The cited references in the second sentence do not directly support the role of auxin in temperature-induced root growth responses. The review by Du et al. (2020) briefly mentions temperature’s effects in the context of general auxin studies but does not focus on cellular growth and not in roots. Gray et al. (1998, PNAS) was probably the first modern study that showed that temperature effects on root growth go via auxin; Zimmermann et al. (2024, PCP) and Yang et al. (2017, PCE), though not centered on auxin, provide stronger evidence of temperature’s effect on root cell growth. Studies specifically linking auxin with root cell growth responses to temperature include Feraru et al. (2019, PNAS) and Ai et al. (2023, EMBO J), which are more relevant than those currently cited.\n• Regarding LLPS condensates, the authors state that these are inactive, which may not be accurate. LLPS condensates can harbor active proteins, depending on their cellular roles, as seen for example in nucleoli, P-bodies, and stress granules.\nMethods:\n• The description of the root cells measured by the authors is unclear. They refer to the region where cell length differences are observed as \"more differentiating cells,\" excluding those \"entering the differentiating phase.\" Are these measurements taken from a narrow zone where cells are still differentiating, or do they also include mature cells? A schematic of a complete Arabidopsis primary seedling root, clearly indicating the region where cells were measured and showing temperature-induced differences in cell length, would be very helpful. Additionally, I strongly recommend measuring cell length in fully mature root cells to clarify whether these temperature-induced changes represent a transient elongation effect without impact on final cell size or whether they significantly influence the length of mature cells.\nMain text:\nFigure 1:\n- When printed, the figure is unreadable (the same applies to all other main figures + several of the Extended Data Figs). I understand that papers are increasingly read on screen, but even then, the reader has to zoom in several times to discern any details. I don’t think figures should be composed in this way. Why not separating the very complex figures into more figures? After all, Nature Communications does not restrict authors to 4 figures only, do they? Additionally, the figures‘ resolution is very low, which may be of course due to compression during the automatic PDF conversion in the submission system.\n- 1A: To which statistical comparison do the asterisks refer to?\n- 1D + l. 54-56: When reporting a correlation between cell length and root length, please include both the quantification and the statistical significance of this correlation. Additionally, it would be valuable to calculate the correlation between root length and cell number. Comparing these correlations could help assess the relative impact of cell division versus cell elongation in temperature-responsive root growth.\n- L. 64: Add plural ‚s‘ to ‚Aux/IAA_ become targeted …‘?\n- 1E-G + l. 69-76: The authors report that several auxin mutants showed cell length defects at the control temperature of 20°C, prior to entering the temperature-responsive growth phase. When quantifying temperature responsiveness (i.e., relative growth at 28°C vs. 20°C), some mutants exhibited a reduced response, which aligns with previous studies. However, I find it concerning that various mutants already displayed temperature-independent defects. For instance, several mutants involved in auxin biosynthesis and signaling (e.g., sav3, yucQ, tir1afb2, saurs) had significantly longer cells even at 20°C. This baseline difference likely dampens their temperature responsiveness, as these mutants would need to elongate much more than WT at 28°C to offset this initial disparity. My question is: How substantial is the defect in temperature responsiveness compared to their general developmental abnormalities, which appear independent of temperature? Put another way, could the authors provide statistical analysis to effectively separate temperature-dependent effects from underlying developmental defects?\n- 1G: Relative data might be skewed because of large differences already under control conditions. Hence, just for themselves, relative data are impossible to interpret. Please provide also absolute cell length data for both temperatures (especially for afb1, arf7 and arf19). And again, absolute root length data are needed for each temperature.\n- 1H: The model illustrating AFB1’s inhibition of TIR1 does not align well with the observed mutant phenotypes, as both afb1-3 and tir1-12 mutants display shorter cells at 28°C compared to Col-0. Although the authors address this in the text accompanying Fig. 3, it remains unclear at this stage, especially since afb1-3 also shows a significant reduction in cell length at 20°C—a point that is not discussed in the manuscript.\nFig. 2:\n- 2A: Scale bar missing + please add the imaged tissue to figure legend.\n- L . 92: ‚downstream of Aux/IAAs‘; same for l. 95\nFig. 4:\n- 4A + l. 131: Is it widely known that increased molecular brightness results from oligomerization? Would it be helpful to include original images, in addition to the quantifications, to illustrate this effect?\n- 4K + l. 168-169: The authors observed that \"warmth reduced the number of meristem cells.\" This appears to be a highly sensitive phenotype, as other studies have reported the opposite effect or no effect at all. For instance, Bin Hamid et al. (2024, https://doi.org/10.1101/2024.05.28.596227) found that higher temperatures actually enlarged root meristem size, while Zhu et al. (2015, Plant Cell Physiol) reported that low temperatures reduce meristem size and cell number. How do the authors account for these conflicting findings across different studies?", "reviewer_2": "Borniego et al. report AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 as novel temperature sensors in the model plant Arabidopsis. The observation that high temperature not only increases auxin biosynthesis, but also nuclear accumulation of AUXIN-SIGNALING F-BOX1 (AFB1), an auxin receptor known to interfere with nuclear auxin signalling, prompted the authors to look for additional temperature-sensitive components downstream of auxin perception. ARF7 and ARF19 are active in the nucleus but can be inactivated by the formation of biomolecular condensates in the cytoplasm. Formation of these condensates is reduced at elevated temperature, resulting in increased nuclear accumulation of these transcription factors and, thus, increased auxin responses. Consequently, an arf7 arf19 double mutant is impaired in temperature-controlled root growth and temperature-enhanced hypocotyl gravitropism.\nThe field of plant temperature responses continues to attract attention and as such, novel temperature input pathways will be of considerable interest for a wider audience. However, there are several discrepancies in the model proposed in the present manuscript that need to be addressed. And while ARF7/19 are shown to play an important role in temperature signalling independent of the known pathways, I have doubts that the data presented fulfil all the criteria for them to be considered “thermosensors”.\nMajor points\n1) Formation of cytoplasmic ARF condensates decreases with increasing temperature from 9 to 34 degrees Celsius (Fig. 4g, h), yet the root growth response changes over this range: cell length increases from 17 to 25 degrees, but then decreases from 25 to 34 degrees (Fig. 4f); arf7 arf19 mutants display shorter cells at 25 and 34 degrees. Which of these responses would be mediated by reduced condensate formation (as it cannot be both)? In the discussion (lines 199-205) the authors also state that de-repression of ARF7/19 via canonical auxin signalling is predicted to inhibit root growth, while directly increased nuclear abundance of ARF7/19 promotes root growth. Why would this be the case? Is there a difference between ARFs de-repressed (due to degradation of Aux/IAA proteins) and ARFs “freshly” imported into the nucleus (due to reduced condensate formation)?\n2) For a molecule to be considered a bona fide thermosensor, it should fulfil three criteria (see Vu et al., 2019, Trends Plant Sci.): (1) Temperature must directly alter its molecular properties; (2) These properties must be important for signalling and (3) affect a physiological or developmental output in a temperature-dependent manner. I note that the companion manuscript (Wilkinson et al.) does not use the term “thermosensors” and care should be taken whether ARF7/19 fulfil all these criteria (based on the data presented).\nTemperature affects formation of ARF molecular condensates, fulfilling criterium 1. However, while ARF7 and ARF19 are involved in auxin signalling (criterium 2) and promote temperature-dependent root growth (criterium 3), it is unclear whether these effects are controlled via condensate formation. The authors mention that a mutation in a conserved lysine residue (K962 in ARF19) disrupts condensate formation, which would be a suitable tool to test criteria 2 and 3, i.e. monitor ARF target gene expression and root growth in this mutant at different temperatures. Power et al. (2019, Mol. Cell) observed that a K962A mutation reduces root length under control temperatures; thus, it seems unlikely that increased root length at mildly elevated temperatures (25-28 degrees) could be a result of reduced condensate formation.\n3) The authors hypothesise that increased nuclear accumulation of AFB1, which is known to antagonise canonical nuclear auxin signalling, attenuates the temperature-induced increase in auxin levels (due to higher biosynthesis). However, TIR1, and to a lesser degree AFB2 and AFB3, also display increased nuclear accumulation at high temperature (Fig. 3a-d). Would this not counteract the effect of AFB1?\n4) Point 3 is related to differences observed between AuxSen, 35S::DII-Venus and DR5::GFP-NLS sensors/reporters. In this context, it is important to note that Feraru et al. (2018, PNAS) found both auxin-sensitive DII and auxin-insensitive mDII constructs used in the auxin input sensor R2D2 to increase in signal at elevated temperature, suggesting this might be an artefact and does not reflect decreased auxin input levels. I do not know whether this also applies to 35S::DII-Venus, but caution should be taken when interpreting these results.\n5) For auxin levels quantified with AuxSen (Fig. 2a), it would be more helpful for the reader if, rather than single nuclei, a ratiometric image of a representative root or root section was shown (as in the original AuxSen paper by Herud-Sikimic et al., 2021, Nature) to better appreciate the variation that exists across the root. Such images can be generated with freely available ImageJ plug-ins (see e.g. Rowe et al., 2022, Methods in Molecular Biology).\n6) The quality of many images is very low, up to a point that makes it hard to appreciate the observations reflected in the quantifications and referred to in the main text. I do not know whether this loss in quality occurred during submission, but higher quality images need to be provided in an updated version of the manuscript.\nMinor points\n7) In general, more information should be provided how fluorescence images were quantified. How were images segmented? Was any background normalization performed?\n8) In the models in Fig. 3h and 4l, a red + sign is used to show protein pools “enhanced by warmth”. However, for both AFB1 and ARF7/19, it applies to proteins in cytoplasmic condensates, free protein in the cytoplasm and protein in the nucleus, while the models described in the text suggest a reduction in cytoplasmic and an increase in nuclear accumulation. This should be reconciled.", "reviewer_3": "Borniego et al reported that ARF7 and ARF19, acting as thermosensors, mediated warmth-induced epidermal cell elongation in root growth. Warmth increased its nuclear abundance and reducing their accumulation in inactive cytoplasmic condensates. This is a very important and interesting study. However, I have a few concerns. And there are two contrary points against previously reports, which needs more discussion and explanation.\n1. The authors hypothesised that the control of ARF7 oligomerisation could be a point of direct temperature input into the system mainly based on previous ITC experiments. However, the temperature dependence of ARF7 oligomerization in the ITC manifests the enthalpic and entropic contributions binding, it is questionable whether this is relevant in vivo. This should be tested in heterologous expression system, e.g. yeast cells, whether PB1 oligomerization is intrinsically sensitive to temperature.\n2. According to Fig 3a, abundant TIR1 was in cytoplasm. However, it has been reported previously that TIR1 was primarily in the nuclear at the same temperature (DOI: 10.7554/eLife.54740). What is the difference?\n3. In many cases, the resolution of the images is low such that it is difficult to see the results described in the manuscript. For example, the authors claimed that warmth increased the nuclear levels of fluorescence driven by pTIR1-TIR1, pAFB2-AFB2 and pAFB3-AFB3 transgenes fused to mCITRINE10 according to Fig. 3a-c. However, it is even not clear to me which signal is from nucleus. All imaging showing nuclear signal should be accompanied by DAPI staining.\n4. Fig 2a: Does temperature affect detection result of AuxSen reporter? It has been known that temperature can affect FRET efficiency. Maybe Fig 2a result does not prove increasing auxin level. And Fig 2b: warm temperature enhanced the fluorescence driven by DII-VENUS, which indicated decreasing auxin level.\n5. Throught the manuscript, the authors proved that warm temperature prolonged the elongation of differentiating cells. However, there seems to be a different opinion that no observe notable temperature-induced differences in cell length throughout the root (DOI: 10.15252/embj.2022111926). Therefore, they should give more proofs and discussion about this.\n6. Line 101, the authors speculated that temperature might reduce the abundance of TIR1/AFB2/AFB3 accounting for the increased DII-VENUS, why?\n7. How do AFB1 or ARF7/19 mutations that block its nuclear import respond to warm temperature? This is an indispensable proof to support the conclusion.\n8. Temperatures higher than the ambient temperature decrease the root length, therefore I wonder how 37 degree or higher temperature affect the distribution of AFB1 and ARF7/19.\n9. reference 39 and 40 are repeated\n10. line 154, change AR19 to ARF19", "reviewer_4": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nOverall evaluation\nBelen Borniego et al. present a detailed analysis of how the auxin co-receptor AFB1 and\nthe two ARFs, ARF7 and ARF19, respond to temperature changes in Arabidopsis\nseedling roots. They describe interesting cellular behaviors suggesting thermosensory\nroles for ARF7 and ARF19 and a repressive effect of AFB1 on nuclear TIR1 signaling. In\nfact, they state that their ‚study identifies ARF7 and ARF19 as bona fide temperature\nsensors essential for root growth‘. I think this central claim of the paper is overstated for\nseveral reasons:\n1. It remains unclear if these cellular phenotypes—intriguing as they are—significantly\nimpact root growth in response to temperature (these essential data are not provided). If\nARF7 and ARF19 sense temperature without markedly affecting root elongation, this\nmay limit the study’s contribution to understanding temperature-induced root growth.", "reviewer_author_response": "RESPONSE TO THE REVIEWER COMMENTS\nReviewer #1\nI appreciate the detailed analysis of cellular growth processes in primary roots presented in Figure\n1, as well as the thoughtful comparison to previously published results. The additional analysis of\ncell length data for auxin-related mutants in Figure 2 (formerly Figure 1) clearly demonstrates the\ncontribution of warmth-induced cell elongation to the root thermomorphogenic response.\nSimilarly, the reporter-based cell biological analyses provide valuable insights into how\nauxin affects differentiating cells. In this regard, the manuscript advances our understanding of the\ncellular mechanisms underlying root thermomorphogenesis in wild-type plants.\nThe central focus of the manuscript, however, is on the role of ARF7 and ARF19 (and to a lesser\nextent AFB1) in warmth-induced root growth promotion. The authors present a thorough analysis\nof cellular growth responses in these mutants, as well as the phase separation behavior of the\nrelevant proteins, which is certainly interesting from a molecular perspective.\nThe main issue, which the authors appear to downplay, is that these molecular phenotypes have\nonly a minimal impact at the organ level. The observed reduction in growth promotion in the\narf7,19 double mutants compared to wild type is only about 3 %. Despite this, the manuscript is\nwritten in a way that suggests the discovery of a central thermosensory phase separation\nmechanism involving ARF7 and ARF19 as key regulators of root thermomorphogenesis. In my view,\nthis is an overstatement intended to lend the results the significance needed for consideration by\na high-impact journal. The claims regarding the physiological relevance of these genes and\nproteins for root thermomorphogenesis are simply not substantiated by the data. As it stands, this\nis a data-rich manuscript about two proteins that appear to have only a minor effect on root\nthermomorphogenesis.\nIn conclusion, I support the publication of this manuscript for its mechanistic detail and the\ninsights it provides into how auxin and ARFs influence cell elongation in the differentiation zone.\nHowever, since the authors frequently use the term 'root growth,' which implies effects at the\norgan level, they should clearly state that these cellular effects translate into only a minimal\nimpact on thermomorphogenesis, with just a 3 % difference observed. The subtle organ growth\nphenotypes should not be obscured, as is currently the case in Figure 2f. Instead, the authors\nshould be transparent and explicitly acknowledge that the overall effect on temperature-induced\nroot growth appears negligible, likely due to buffering processes that remain to be elucidated.\nIt is important for readers to understand that ARF7 and ARF19 are not thermosensors that\nregulate temperature-induced growth to the same extent as PhyB or ELF3 do in the shoot, where\nloss-of-function mutants exhibit pronounced hypocotyl growth phenotypes. The likely presence of\nbuffering mechanisms mitigates the cellular effects described in this study.\nOverall, while the data clearly demonstrate robust cellular responses to elevated temperature,\nthese effects are substantially buffered at the organ level, resulting in only minimal changes in\noverall root growth. This suggests that, although the underlying molecular and cellular\nmechanisms are active and mechanistically significant, additional regulatory or compensatory\nprocesses at higher organizational levels mitigate their impact on organ-scale phenotypes.\nUnderstanding these buffering mechanisms is an important avenue for future research and does\nnot diminish the value of elucidating the cellular pathways involved."}, "author_response": ""}]

Decision letter