bZIP factors of the Unfolded Protein Response interact with PIF4 to promote thermomorphogenesis

Domain: biology
Published: 2025-12-30
Editorial outcome: major_revision

Abstract
When Arabidopsis plants are exposed to warm temperatures (e.g., 29°C), they undergo adaptive growth known as thermomorphogenesis. This process is primarily regulated by the phytochrome B (phyB)-PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) module; however, the potential involvement of additional signaling pathways remains underexplored. Here, we show that warmth triggers endoplasmic reticulum (ER) stress, activating both arms of the Unfolded Protein Response (UPR). Three UPR-associated bZIP transcription factors, bZIP17, bZIP28 and bZIP60, promote hypocotyl growth under warmth in a PIF4-dependent manner. Active bZIP factors form complexes with PIF4 in the nucleus, where they bind to promoter regions of PIF4 and other growth-related genes to enhance their expression. In parallel, bZIPs overexpression counteract the inhibitory effect of phyB on PIF4 stability, thereby reinforcing thermomorphogenic growth. Together, our findings define a regulatory axis that links ER stress, the UPR and thermomorphogenesis, clarifying how plants coordinate physiological and environmental cues to adapt to warming conditions.

Introduction
Plants are constantly being exposed to environmental stressors that disrupt cellular functions leading to impaired growth and reduced productivity1–3. One essential mechanism for mitigating stress-induced damage is the Unfolded Protein Response (UPR), a conserved signaling pathway that alleviates endoplasmic reticulum (ER) stress caused by the accumulation of misfolded proteins under stress conditions4,5. In plants, the UPR is primarily regulated by three BASIC LEUCINE ZIPPER (bZIP) transcription factors, bZIP17, bZIP28, and bZIP60. In response to ER stress, bZIP17 and 28 undergo proteolytic processing, while bZIP60 mRNA undergoes IRE1-dependent unconventional splicing, leading to the generation of active forms of bZIP factors–bZIP17N, bZIP28N, and bZIP60s, which translocate to the nucleus to regulate expression of stress-responsive genes4–7.

Heat stress is a well-known inducer of ER stress, disrupting protein homeostasis and, subsequently, activating the UPR8–13. Priming-mediated thermotolerance at 37°C further demonstrates the involvement of UPR components14; however, whether moderately elevated temperatures also induce ER stress remains unclear. Thermomorphogenesis, an adaptive growth response to moderately elevated temperatures (e.g., 29°C), is primarily regulated by PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), which controls the expression of growth-related genes15–17. Given that thermomorphogenesis requires increased synthesis of secreted proteins for cell elongation, moderate temperature stress may impose additional pressure on the ER, raising the possibility that UPR signaling may contribute to thermomorphogenesis.

Results
Warm temperature rapidly activates UPR signaling prior to induction of canonical thermoresponsive genes
To determine whether elevated ambient growth temperatures would trigger UPR, we exposed Arabidopsis seedlings to 29°C (warmth) and analyzed gene expression levels at multiple time intervals. Figure 1a shows that in a short-term kinetic assay, bZIP28 transcripts increased within 20 min upon warmth treatment, followed by the elevated expression of UPR marker BiP1 at 30 min (Fig. 1b). PIF4 transcripts were subsequently induced at 50 min, with activation of the PIF4 target gene YUCCA8 (YUC8) observed at a later time point, 60 min (Fig. 1c). In support of these findings, analysis of a previously published RNA-seq dataset revealed that bZIP28 transcript abundance also increased within 20–30 min of warm treatment, whereas PIF4 induction was detected only after 30 min18, consistent with the temporal pattern we observed. These results indicate that UPR signaling is engaged earlier than the induction of canonical thermoresponsive genes during the transition to warm conditions.

To assess whether UPR activation is maintained over longer periods, transcript levels were monitored at extended time points (5 h to 13 d; Supplementary Fig. 1a). BiP1 and bZIP28 transcripts remained elevated under prolonged warmth, and spliced bZIP60 (bZIP60s) transcripts were also induced, indicating activation of the second major UPR signaling branch (Supplementary Fig. 1b). This observation is reminiscent of that found with PIF4 and YUCCA8 (YUC8) genes (Supplementary Fig. 1c). Notably, the active nuclear form of bZIP28 (bZIP28N) was clearly detected by immunoblotting after 2 days of warmth treatment, but not under normal conditions (Supplementary Fig. 1d). Collectively, these findings demonstrate that warm temperature induce ER stress, rapidly activates UPR signaling ahead of PIF4 and YUC8 induction, and that this activation is sustained during prolonged exposure, suggesting an early involvement of UPR in thermomorphogenic growth.

Mild pharmacological ER stress promotes hypocotyl elongation in a PIF4-dependent manner
Activation of UPR signaling induced by pharmacological agents, including treatment with tunicamycin (Tm) and dithiothreitol (DTT), often results in strong seedling growth arrest and chlorosis, possibly due to drug overdose, which is in stark contrast to the growth-promoting effects of thermomorphogenesis. We therefore examined the effects of low Tm dosage on hypocotyl elongation, which is the most prominent and widely used marker for seedlings thermomorphogenesis. Arabidopsis seedlings were treated with 8 μg/L Tm, 10 times lower than the working concentration commonly used to induce acute ER stress (Supplementary Fig. 2a). Intriguingly, at this mild dose, seedlings displayed a modest but statistically significant increase in hypocotyl lengths at 22°C, and such effect was further enhanced at 29°C (Supplementary Fig. 2b, c). BiP1 transcripts accumulated in Tm-treated seedlings compared to DMSO controls at 0 and 2 d under both temperature conditions, while bZIP28 expression was significantly upregulated at 1 d under normal condition, and 2 d under warmth (Supplementary Fig. 2d, e). Notably, compared to normal temperature condition, BiP1 and bZIP28 transcripts accumulation increased at 29°C, further supporting our observations that elevated temperature enhances ER stress. Together with the longer hypocotyls observed in Tm-treated seedlings at 29°C relative to 22°C, these results suggest a functional interplay between UPR signaling and thermomorphogenesis.

Since PIF4 functions as a central regulator of plant thermomorphogenesis through activating warmth-responsive gene expression, we examined its involvement in UPR-mediated growth regulation. Hypocotyl elongation induced by modest Tm treatment was abolished in pif4 seedlings at either 22°C or 29°C (Supplementary Fig. 2b, c), indicating that Tm-induced UPR promotes hypocotyl elongation in a PIF4-dependent manner.

To determine whether PIF4 itself is involved in UPR regulation, we examined ER stress response of pif4. No significant differences in shoot or root growth were observed between pif4 and WT seedlings upon Tm treatment (Supplementary Fig. 3a, b). Furthermore, analysis of UPR marker genes (BiP1 and BiP3) showed comparable transcriptional response to Tm treatment between pif4 and WT seedlings (Supplementary Fig. 3c). These results exclude the possibility of a defective UPR signalling in pif4.

Next, we examined whether the warmth-induced transcriptional activation of UPR signaling is regulated by PIF4. Notably, whereas pif4 was completely insensitive to warmth treatment in terms of hypocotyl elongation, the transcriptional upregulation of BiP1 and bZIP28 in pif4 was similar to that in WT seedlings under both short-term and prolonged warm treatments (Supplementary Fig. 4a, b). This finding suggests that warmth-induced activation of UPR signaling is independent of PIF4 regulation and likely functions upstream of PIF4 signaling.

bZIP factors of UPR promote thermomorphogenesis
Given that UPR-associated bZIPs are transcriptionally responsive to warmth, we next examined their roles in thermomorphogenesis. Single knockout mutants of bzip17, bzip28, bzip60 exhibited no significant differences in hypocotyl lengths compared to WT at either 22°C or 29°C (Fig. 2a, b). Remarkably, at warmth, double mutants bzip17/60 and bzip28/60 displayed significantly shorter hypocotyl compared to WT, whereas remaining indistinguishable from WT at 22°C (Fig. 2a, b). This suggests that the bZIP factors derived from both arms of the UPR pathway function alone or in concert with other factors to promote plant thermomorphogenic hypocotyl growth. In line with impaired hypocotyl elongation induced by warmth, YUC8 and PIF4 transcript levels were reduced in bzip17/60 and bzip28/60 double mutants compared to WT (Fig. 2c, d).

To further corroborate the positive roles of bZIP17, bZIP28 and bZIP60 in regulating thermomorphogenesis, we generated gain-of-function mutants overexpressing either the full-length or nuclear form of individual bZIPs in WT background. Compared to WT, both full-length and nuclear bZIPs-OE displayed enhanced hypocotyl elongation under warmth (Supplementary Fig. 5a, b, and 6, and Fig. 2e, f). In line with previously observed defects in thermomorphogenic hypocotyl growth in bzip loss-of-function mutants, warmth-induced hypocotyl elongation and marker gene expression were significantly enhanced in overexpression lines with no observable growth alteration at 22°C (Fig. 2g, h).

The difference in hypocotyl lengths between bzip17/60, bzip28/60 double mutants and pif4 prompted us to further investigate the functional relationship between bZIPs and PIF4 during thermomorphogenesis. To this end, we crossed the double mutants with pif4. At warmth, the intermediate hypocotyl lengths of the double mutants were reduced to those of pif4 in the bzip17/60/pif4 and bzip28/60/pif4 triple mutants (Fig. 2a, b). We further generated bZIP OE lines in pif4 mutant background by crossing bZIP17N-OE, bZIP28N-OE or bZIP60s-OE lines with pif4. Western blot analysis confirmed bZIP protein levels in the OE/pif4 lines were comparable to those in the WT background (Supplementary Fig. 6d). Interestingly, compared to the bZIP overexpression in WT background, hypocotyl elongation in the OE/pif4 lines were insensitive to warmth, a phenotype similar to that of pif4 (Fig. 2g, h). Together, these results indicate that PIF4 is required for bZIP-mediated promotion of thermomorphogenesis.

To further corroborate these observations, we generated double overexpression lines (bZIP28N/60s-OE and bZIP17N/60s-OE) in both WT and pif4 background by genetic crosses. Neither bZIP28N/60s-OE/WT nor bZIP17N/60s-OE/WT displayed additional increased hypocotyl elongation compared to the single bZIP-OE line. These results suggest that bZIP17, bZIP28 and bZIP60 function in a shared pathway to regulate thermomorphogenesis (Supplementary Fig. 5c, d). Furthermore, bZIP28N/60s-OE/pif4 and bZIP17N/60s-OE/pif4 failed to rescue the pif4 growth defect, confirming that UPR-mediated regulation of thermomorphogenesis is PIF4-dependent.

bZIP active forms, bZIP17N, bZIP28N and bZIP60s, interact with PIF4 in the nucleus and bZIP/PIF4 complexes are bound to promoter regions of PIF4-target genes
Since the thermomorphogenic growth mediated by UPR-bZIPs requires PIF4 functions, we hypothesized that the bZIP factors might function in a protein complex with PIF4 to regulate expression of downstream growth-promoting genes. Under ER stress conditions, the N-terminal domains of bZIP17 and bZIP28, along with bZIP60s, are translocated to the nucleus. We observed that the active forms of bZIPs co-localized with PIF4 in nuclei when transiently overexpressed in Nicotiana benthamiana (hereafter Nicotiana, Supplementary Fig. 7a). Quantitative colocalization analysis further supported this overlap, with Pearson correlation coefficients of ~0.5, indicating a nuclear co-localization between bZIP active forms and PIF4 (Supplementary Fig. 7b).

To examine whether there are direct interactions between bZIPs and PIF4, we performed in vitro pull-down assays using recombinant maltose binding protein (MBP)-tagged PIF4 and mCherry-tagged bZIP active forms. We found that PIF4 directly interacted with bZIP17N, bZIP28N, and bZIP60s but not with the mCherry negative control (Fig. 3a). In vivo associations were confirmed by co-immunoprecipitation (Co-IP) assays, wherein PIF4-flag co-immunoprecipitated with bZIP17N-HA and bZIP28N-HA, while PIF4-myc was co-immunoprecipitated by bZIP60s-flag in planta (Fig. 3b). In addition, PIF4-myc also co-immunoprecipitated with bZIP17N-HA, bZIP28N-HA (Supplementary Fig. 8). Intriguingly, we observed similar patterns under warmth condition, indicating that PIF4-bZIP complexes are preformed and maintained upon warming (Fig. 3b and Supplementary Fig. 8). In addition, results of Bimolecular fluorescence complementation (BiFC) assays in Nicotiana suggest that the active form of bZIPs interact with PIF4 in nuclei. Strong GFP fluorescence was detected in nuclei when PIF4-GFPC was co-expressed with GFPN-bZIP17N, GFPN-bZIP28N, or GFPN-bZIP60s whereas no signal was detected with the free GFPN (Fig. 3c). Taken together, these results suggest that the active forms of UPR-associated bZIPs directly interact with PIF4 in Arabidopsis nuclei. This finding is consistent with previous reports showing that bZIP transcription factors interact with bHLH proteins19,20.

Under warmth conditions, PIF4 directly targets multiple growth-promoting genes including PIF4 itself to regulate thermomorphogenic growth17,21. To investigate whether bZIPs also bind to the promoter regions of PIF4-targeted warmth-responsive genes, we performed electrophoretic mobility shift assays (EMSA) using a DNA probe from the PIF4 promoter region. bZIP17N, bZIP28N, bZIP60s proteins specifically interacted with the G-box element within the PIF4 promoter, forming distinct protein-DNA complexes (Fig. 3d). These associations were strongly reduced with the addition of a 100-fold molar excess of non-labeled G-box probes as a competitor and bindings were abolished when the G-box probes were mutated, consistent with previous reports that bZIP transcription factors can bind G-box elements in gene promoters17,22. These results indicate that bZIP proteins directly associate with the PIF4 promoter region.

To confirm the binding in vivo, we performed chromatin immunoprecipitation (ChIP)-qPCR assays. bZIP17N, bZIP28N and bZIP60s were specifically enriched at the promoter regions of various PIF4-targeted genes, PIF4, YUC8, and IAA19 (Fig. 3e–g). Although a weak enrichment of bZIPs at target promoters were detectable at 22°C compared with the WT control, this basal binding was consistent with our EMSA results. Notably, such binding was markedly stronger at 29°C, indicating that bZIPs association with these promoters is specifically enhanced under warmth.

Consistent with the observation that thermomorphogenic growth promoted by UPR-associated bZIPs requires PIF4 functions, the association of bZIPs with promoters of PIF4 target genes was significantly reduced and failed to display the warmth-induced binding activity in the absence of PIF4 (Fig. 3e–g). These results suggest that PIF4 is essential for the recruitment of bZIPs to the target gene promoters. Collectively, these findings indicate that bZIPs and PIF4 form a regulatory complex to orchestrate expression of growth-promoting genes during thermomorphogenesis.

bZIP factors enhance PIF4 protein stability during thermomorphogenesis
PIF4 function is regulated by temperature at both transcriptional and posttranslational levels, and PIF4 protein stability is crucial for modulating warmth response of plants17. We found that warmth-induced transcriptional upregulation of PIF4 was significantly reduced in bzip mutants, correlating with a strong reduction in PIF4 levels upon warm treatment (Supplementary Fig. 9a). In line with the enhanced thermomorphogenic growth observed in bZIP overexpression plants, PIF4 transcript levels were highly upregulated when active form of UPR-associated bZIPs were overexpressed resulting in a greater PIF4 protein abundance (Supplementary Fig. 9b).

To determine whether bZIPs regulate PIF4 beyond modulating its transcript levels, we overexpressed PIF4-HA under a 35S promoter in WT, bzip17/60 and bzip28/60 mutant backgrounds (Fig. 4a, b). Plants with comparable PIF4 transcript levels were selected for further analysis (Fig. 4c). We found that PIF4-HA protein accumulation was impaired in bzip mutants as compared to WT under normal growth condition; nevertheless, the difference became more pronounced when plants were subjected to warmth treatment (Fig. 4d). Consistent with the reduced PIF4 levels, we also observed shorter hypocotyl lengths in bzip double mutant background as compared to WT.

To explore the roles of bZIPs in regulating PIF4 protein stability, we treated transgenic lines with cycloheximide (CHX) to block de novo protein synthesis and monitored PIF4 degradation over time. Upon CHX treatments, PIF4-HA degradation was accelerated in the bzip28/60 mutants compared with WT under both normal and elevated temperatures (Fig. 4e, f). These results demonstrate that bZIPs are required to stabilize PIF4 protein in vivo. Moreover, the effect of bZIP28 and bZIP60 on PIF4 stability implicate these two major UPR regulators are collectively involved in thermomorphogenic signaling.

Additionally, we examined whether bZIPs influence PIF4 transcriptional activity. To ensure comparability, equivalent amount of PIF4-HA protein was used for ChIP assays. We found comparable binding of PIF4-HA to its target promoters (pPIF4, pYUC8 and pIAA19) in both WT and bzip double mutants (Supplementary Fig. 10). These results suggest that the absence of bZIPs does not impair transcriptional activity of PIF4 but instead it reduces PIF4 protein stability thereby limiting its ability to promote hypocotyl elongation.

bZIP factors partially offsets phyB-mediated inhibition of PIF4 function
phyB is a red/far-red light photoreceptor that also functions as a thermosensor in Arabidopsis. At elevated temperatures, phyB undergoes thermal reversion to its inactive state, thereby releasing its suppression of PIF4 and enabling thermomorphogenic growth program23,24.

To investigate the interplay between bZIPs and the PIF4-phyB module, we examined UPR activation in phyB mutant and 35S::YHB plants, wherein the later expresses a constitutively active phyB variant (YHB, carrying a Y276H substitution) under warm condition. qPCR analysis suggested a wild-type level of bZIP28 and BiP1 expression in both backgrounds, indicating that phyB does not affect UPR activation under warm conditions (Supplementary Fig. 11).

We then assessed the impact of bZIP28 and bZIP60 on PIF4 stability in the context of phyB regulation. Thermomorphogenic responses were strongly suppressed in 35S::YHB plants, whereas overexpression of bZIP28N or bZIP60s partially restored elongation at elevated temperature (Fig. 5a, b and Supplementary Fig. 12a). Consistently, increased PIF4 protein levels were detected in bZIP28N/35S::YHB and bZIP60s/35S::YHB seedlings compared with 35S::YHB alone, and the repression of YUC8 by YHB was alleviated in these lines (Supplementary Fig. 12b, c). These results together demonstrate that bZIP28N and bZIP60s counteract the inhibitory effect of phyB on PIF4 accumulation and function under warm condition.

Pull-down and BiFC assays showed that bZIP17N, bZIP28N, and bZIP60s directly interact with phyB in vitro and in vivo (Fig. 5c, d). To test whether bZIPs affect the phyB-PIF4 interaction in planta, we performed firefly luciferase complementation imaging (LCI) assays in Nicotiana. Co-expression of PIF4-cLuc and phyB-nLuc generated a robust luciferase signal, confirming their in vivo interactions. Notably, co-expression of bZIP28N-YFP or bZIP60s-YFP strongly reduced the PIF4-phyB interaction (Fig. 5e, f). These findings indicate that the major UPR regulators bZIP28N and bZIP60s stabilize PIF4 by competitively attenuating its interaction with phyB, thereby promoting PIF4 function under warm conditions.

Previous work showed that HSFA1s stabilize PIF4 during thermomorphogenesis without affecting its DNA-binding capacity25. Given that UPR-associated bZIPs also stabilize PIF4, we asked whether these pathways might intersect. Pull-down assays revealed that bZIPs directly interact with HSFA1d, but such interactions are independent of PIF4 (Supplementary Fig. 13a). Moreover, in the hsfa1 quadruple mutant, bZIPs still interact with PIF4, indicating that their association is HSFA1-independent (Supplementary Fig. 13b). We further interrogated published RNA-seq data of short-term warm-treatment and found that transcripts of HSFA1 accumulate later than those of bZIP28 and PIF418. Together, these observations imply that bZIPs and HSFA1s may act on the regulation of PIF4 stability in a sequential order, wherein bZIPs contribute to early stabilization of PIF4, which is further reinforced by HFSA1s during prolonged warming possibly in a joint effort with bZIPs. Future studies are therefore required to dissect the underlying molecular mechanism in detail.

Methods
Plant materials and construction of transgenic plants
Wild-type (WT) and mutant plants of Arabidopsis thaliana (hereafter Arabidopsis; Columbia-0, Col-0 ecotype) were used in this study. The T-DNA insertion mutants bzip17 (SALK_104326), bzip28 (SALK_132285C), bzip60 (SALK_050203), and pif4 (CS66043) were obtained from the Arabidopsis Biological Resource Center (ABRC). Double mutants bzip17/60 and bzip28/60 were obtained from Prof. Jeanmarie Verchot32. Triple mutants bzip17/60/pif4 and bzip28/60/pif4 were generated by genetic crosses. The hsfa1-cq mutant was obtained from Prof. Wenqiang Tang’s laboratory.

To generate full-length bZIP17, bZIP28 and bZIP60 overexpression lines in the WT background, the coding sequences of these genes were cloned into pBA-GFPN-DC vector via the Gateway system to produce 35S::GFPN-bZIPs.

For overexpression of the N-terminal fragments bZIP17N and bZIP28N in WT background, DNA sequences encoding the N-terminal fragment of bZIP17 (amino acids 1 to 366, 1098 bp) and bZIP28 (amino acid 1 to 323, 969 bp) were cloned into pBA-DC-HA vector using the Gateway system (Invitrogen, US). The resulting constructs were 35S::bZIP17N-HA (bZIP17N-OE) and 35S::bZIP28N-HA (bZIP28N-OE), respectively. For bZIP60s overexpression in WT, DNA sequences encoding the spliced form of bZIP60 were synthesized by BioBasic Company (Canada) and cloned into the pUBQ-DC-flag vector to generate UBQ::bZIP60s-flag (bZIP60s-OE).

These constructs were used to generate transgenic bZIPs-OE lines, which were then crossed with the pif4 mutant to obtain bZIPs-OE/pif4 seedlings.

For PIF4 overexpression in the WT, bzip17/60, or bzip28/60 double mutant, the full-length PIF4 coding sequence was cloned into pUBQ-DC-flag, pBA-DC-myc or pBCO-DC-HA vectors to generate UBQ::PIF4-flag, 35S::PIF4-myc or 35S::PIF4-HA, respectively. These constructs were introduced into Arabidopsis through Agrobacterium tumefaciens (GV3101)-mediated floral dipping transformation33. Double transgenic plants, including bZIP17N-HA/PIF4-myc, bZIP28N-HA/PIF4-myc, bZIP17N-HA/PIF4-flag, bZIP28N-HA/PIF4-flag and bZIP60s-flag/PIF4-myc, bZIP17N/60s-OE, bZIP28N/60s-OE in both WT and pif4 background, were obtained by genetic crosses.

For YHB overexpression in the WT, bZIP28N-OE, and bZIP60s-OE seedlings, the full-length phyB coding sequence was first cloned into pENTY vector. A site-directed mutation (Y276H substitution) was then introduced to generate YHB, which was subsequently cloned into pBA-DC-myc vector to generate 35S::YHB-myc.

Plant growth conditions and treatments
Arabidopsis and Nicotiana benthamiana (hereafter Nicotiana) plants were grown in a greenhouse at 22°C under long-day conditions (16-h light/8-h dark). Seeds were surface sterilized for 10 min in 70% (v/v) ethanol, followed by a wash in 98% (v/v) ethanol and let dry on a sterile bench. Seeds were sown on half-strength Murashige and Skoog (MS) media solidified with 0.8% agar (w/v) and supplemented with 1% sucrose (w/v). The seeds were stratified at 4°C for 2 days in the dark and then transferred to a chamber with LED light illumination (70 μmol m−2 s−1) under 16 h light/8 h dark.

For the time course experiment at 29°C, seedlings were grown at 22°C for 3 days before being transferred to 29°C or kept at 22°C for 10, 20, 30, 40, 50, 60, 120, 180, 240 min, 5 h, 1, 2, 3, 4, 5, 6, 7, 9, 11, and 13 additional days. Samples taken at the indicated time point were frozen in liquid nitrogen.

For hypocotyl elongation measurements, mutant seedlings were grown on plates at 22°C for 3 days and then transferred to 29°C or kept at 22°C for 4 additional days. Overexpression seedlings were grown at 22°C for 2 days and transferred to 29°C or kept at 22°C for 3 additional days. After the treatment period, images of seedlings were taken and hypocotyl lengths were measured using ImageJ software.

Seedling grown at 22°C for 7 days were used for analysis of transcript and protein levels. Seedlings were either maintained at 22°C or transferred to 29°C for an additional 6 h (for mutant seedlings) or 3 h (for overexpression seedlings). Samples were frozen in liquid nitrogen.

RNA expression analysis
Total RNAs were extracted using EasyPure Plant RNA Kit (Transgen, China). Reverse transcription (RT) was performed using 1 μg RNA and PrimeScriptTM RT Master Mix (Takara, Japan) following the manufacturer’s instructions. The cDNA solution was diluted (1:2) with nuclease-free water. Quantitative PCR (qPCR) was performed using SYBR Green Master (Bio-Rad, USA) on Bio-Rad CFX96 real-time system. Expression levels of target genes were normalized by actin transcript levels. Semi-quantitative PCR were performed using specific primers to detect transgenic PIF4 cassette. All primers are listed in Supplementary Table 1. All experiments were performed with at least three independent biological replicates.

Immunoblotting
Seedlings were homogenized in liquid nitrogen, and lithium dodecyl sulfate (LDS) buffer was added at a ratio of 1:3 (fresh weight: buffer). Protein samples were boiled for 10 min and separated on 10% SDS-PAGE gels. Gels were transferred to 0.45 μm PVDF membranes (Merck, USA), followed by incubation in blocking buffer (1X TBS buffer including 0.1% Tween 20 and 5% dried nonfat milk) for 1 h at room temperature. Membranes were then incubated overnight at 4°C with anti-PIF4 (Agrisera, Cat no. AS163955; 1:3000), anti-myc (Santa Cruz, sc-40; 1:3000), anti-HA (Santa Cruz, sc-7392X; 1:3000) or anti-ACTIN (Proteintech, 60008-1-Ig; 1:5000). Afterward, membranes were incubated for 1 h with a peroxidase-conjugated secondary antibody: anti-mouse (cytiva, NXA931; 1:5000), anti-rabbit (cytiva, NA934; 1:5000) or anti-Goat (Agrisera, AS09605: 1:2000). ACTIN levels, measured by Western blots and the Coomassie blue stained gel band of RbcL (RUBISCO large subunit) were used as loading controls. Fluorescent signals were captured on iBright (Invitrogen, USA).

Protein expression and purification
The coding sequences of bZIP17N, bZIP28N, and bZIP60s were cloned into pET28a-mCherry to generate His-bZIP-mCherry constructs. The CDS of PIF4 was cloned into pET28a to generate His-PIF4 construct. The CDS of HSFA1d, N-, or C-terminal of phyB, PIF4 was cloned into pMAL-DC vector to generate MBP-HSFA1d, MBP-phyBN, MBP-phyBC, and MBP-PIF4 construct, respectively. Protein expression constructs were transformed into Escherichia coli strain Rosetta (Novagen/Merck, Darmstadt, Germany), and recombinant proteins were expressed and purified as described previously34. Briefly, induced cells were suspended in lysis buffer (50 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM MgCl2, 1 mg/ml lysozyme, 10% Glycerol) and gently shaken for 30 min in a cold room. Bacterial cells were lysed by sonication (Qsonica, Newtown, CT, USA) on ice at 40% amplitude, 15 s on/30 s off, 15 cycles, and Triton X-100 was then added to a final concentration of 1%. Cell lysates were centrifuged at 21,000 × g for 1 h (Beckman Coulter, Pasadena, CA, USA). The supernatant was mixed with equilibrated amylose (NEB, Ipswich, MA, USA) or Ni-NTA (Bio Basic Inc. Canada) beads, and protein purification was carried out according to the manufacturers’ instructions. Protein purity was assessed by SDS-PAGE.

In vitro pull-down assays
500 ng of purified proteins were incubated in reaction buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.2% glycerol, and protease inhibitor (Roche)) for 1 h at room temperature (RT). Equilibrated resins were added to the mixture, which was then further incubated for 1 h at RT. The resins with bound proteins were washed 5 times with the reaction buffer. Bound proteins were eluted by LDS and incubated at 98°C for 5 min. Proteins in samples were separated by SDS-PAGE followed by Western blot analysis using anti-MBP (Proteintech, Cat no. 66003-1-Ig; 1:3000) and anti-mCherry (Proteintech, Cat no. 26765-1-AP; 1:3000).

Electrophoretic mobility shift assay (EMSA)
EMSA were performed using Biotin 3’ End DNA Labeling Kit and Lightshift Chemiluminescent EMSA kit according to the manufacturer’s instructions (Thermo Scientific). MBP, MBP-bZIP28N, -bZIP17N, mCherry, mCherry-bZIP60s recombinant proteins were incubated in a 20 μl reaction mixture containing 100 mM Tris-HCl pH 8.0, 50 mM KCl, 5 mM MgCl2, 0.05% NP-40, 1 mM EDTA, 50 ng/ml poly (dI-dC) for 10 min at room temperature. The biotin-labeled probes were then added to the mixture and incubated for additional 20 min at room temperature before separation on 4% native polyacrylamide gels in 0.5X TBE buffer. The labeled probes were detected with streptavidin according to the instructions provided by the manufacturer (Pierce).

Chromatin immunoprecipitation (ChIP) assays
ChIP experiments were performed as described previously35. Ten-day-old WT, bZIP17N-OE/WT, bZIP17N-OE/pif4, bZIP28N-OE/WT, bZIP28N-OE/pif4, bZIP60s-OE/WT and bZIP60s-OE/pif4 seedlings, transferred to 29°C for 5 h or kept at 22°C, were used for ChIP assays. Approximately 2 g of each sample was harvested and cross-linked in 1% formaldehyde for 15 min, followed by 5-min neutralization with 0.125 M glycine. After washing 6 times with distilled water, seedlings were ground into powder in liquid nitrogen. Chromatin complexes were isolated and sonicated. The specific chromatin complex was then immunoprecipitated using anti-HA (Santa Cruz Antibodies, Cat no. sc-7392X; 1:3000) or anti-flag antibody (lab-made). The precipitated DNA was recovered and analyzed by real-time qPCR using the respective primer pairs listed in Supplementary Table 1.

Agrobacterium-mediated transient overexpression
For the co-localization experiment, the coding sequences of bZIP17N, bZIP28N and bZIP60s were cloned into pBA-DC-GFP, and YHB or PIF4 was cloned into pBA-DC-CFP. For the BiFC assay, the coding sequences of bZIP17N, bZIP28N and bZIP60s were cloned into the pBA-GFPN-DC, and PIF4 was cloned into pBA-DC-GFPC using Gateway system. These binary vectors were transformed into Agrobacterial GV3101, and colony was inoculated into liquid LB medium and grown overnight. Harvested cells were resuspended in the Agrobacterium resuspension buffer (10 mM MgCl2, 10 mM MES and 0.2 mM acetosyringone) to an optical density of ~1.0 at 600 nm (OD600) and allowed to sit for 2–3 h. Nicotiana leaves were then infiltrated with the bacterial suspension using a 1-mL syringe without a needle. Leaves were examined for fluorescence signal at 2 days post agroinfiltration by Confocal microscopy (FV3000 confocal laser scanning microscope, Olympus, Japan). Excitation wavelengths used were 488 nm for GFP and 406 nm for CFP. Pearson’s correlation coefficient (PCC) was calculated using CellSens software based on confocal images acquired with the FV3000 microscope.

Co-immunoprecipitation assays
Transgenic plants expressing bZIP17N, bZIP28N, bZIP60s, PIF4 or their combinations were used to detect possible interactions. Seedlings were grown in a chamber at 22°C under long-day conditions for 7 days before being transferred to 29°C for 5 h. Harvested samples were ground into a fine powder in liquid nitrogen and homogenized in 2X IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 5% Glycerol, 50 μM MG132, protease inhibitor cocktail tablets (Roche)). Extracts were centrifuged two times at 12,000 × g for 20 min. The supernatant was mixed with 40 μl anti-myc magnetic beads (for bZIP28N and PIF4 interaction test) (Santa Cruz, sc-500772) or anti-flag M2 magnetic beads (Merck, Cat no. M8823) for 4 h at 4°. The beads were then washed 5 times with 1X IP buffer. Samples were boiled in SDS-PAGE sample buffer, and the co-immunoprecipitated proteins were detected with anti-HA, anti-flag or anti-myc antibody (Cell Signaling Technology, Cat no. #2276; 1:3000).

ER stress treatment and phenotypic analysis
Experiments with Tunicamycin (Tm) were performed by growing seedlings directly on plates containing various concentrations (8, 40, 60 and 80 μg/L) of Tm (Sigma-Aldrich, Cat no. T7765). Tm was replaced by the same volume of dimethyl sulfoxide (DMSO) in control treatment. After 3 days at 22°C, seedlings on 8 μg/L Tm or DMSO plates were transferred to 29°C or kept at 22°C for 4 additional days. Seedlings images were captured and hypocotyl lengths were measured by ImagJ. For normal phenotypic analysis of ER stress, shoot (grown horizontally) and primary root growth (grown vertically) of 7d-old seedlings were captured. Root lengths were measured by ImageJ software. For qRT-PCR analyzes, seedlings were grown on half MS medium with 1% sucrose and 0.8% agar for 10 days, followed by transfer to 5 μg/ml Tm plates for 5 h. All data shown are the mean of at least 3 independent biological experiments.

Protein degradation assessments
10-day-old seedlings of PIF4-HA/WT, PIF4-HA/bzip17/60 and PIF4-HA/bzip28/60 were transferred to MS liquid medium and incubated overnight. To examine the half-life of PIF4-HA proteins in these genotypes, seedlings were treated with 50 μM MG132 for 6 h to block proteasome activity. After washing three times, seedlings were transferred to a medium containing 200 μM cycloheximide (Sigma-Aldrich, Cat no. C1988) to stop new protein synthesis. Samples were harvested at different time points, and protein extracts were analyzed by immunoblotting.

Firefly LCI assays
The LCI assays for the protein interaction detection were performed in N. benthamiana leaves as described previously36–38. Briefly, the full-length of the genes was cloned into pCAMBIA1300-nLuc or pCAMBIA1300-cLuc vectors, respectively. These vector constructs and control vectors were transformed into Agrobacterium strain GV3101. The bacteria that contain nLuc or cLuc constructs were cultured overnight and then mixed in equal ratios. The bacterial mixtures were then introduced into N. benthamiana leaves using a needle-less syringe. Before imaging, the leaves were infiltrated with 1 mM D-luciferin and then kept in the dark for 5 min. The LUC activities were analyzed after 48-h infiltration using Retiga LUMO CCD (Teledyne, Canada).

Accession numbers
For Arabidopsis genes, accession numbers given by the Arabidopsis Information Resource (https://www.arabidopsis.org/) are indicated. bZIP17 (AT2G40950), bZIP28 (AT3G10800), bZIP60 (AT1G42990), PIF4 (AT2G43010), actin (AT3G18780), BiP1 (AT5G28540), BiP3 (AT1G09080), YUC8 (AT4G28720), IAA19 (AT3G15540), PP2A (AT1G69960), phyB (AT2G18790), HSFA1d (AT1G32330).

Statistical analyses
Data are presented as mean ± standard deviation, with error bars indicating the standard deviation. Comparisons between two groups were performed using a two-tailed Student’s t-test in Microsoft Excel 2016. P < 0.05 considered statistically significant, and P < 0.01 highly significant. Comparisons among more than two groups were conducted using one-way ANOVA followed by Tukey’s post hoc test, performing using the Statistics Kingdom online tool.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the manuscript \"bZIP factors of the Unfolded Protein Response interact with PIF4 to promote thermomorphogenesis”\nZhang and colleagues investigate the mechanisms that regulate heat stress by activation of different response pathways.\nSpecifically, the study focuses on the cross-talk between two pathways: the thermomorphogenic response induced by\nmoderate temperature increases and the unfolded protein response.\nAlthough the role of the UPR in high-temperature heat stress is well established, the triggering of this pathway, and the role\nof its main players, in the response to moderate temperatures (mild heat stress), is not yet fully elucidated.\nBecause thermomorphogenesis is a protective mechanism induced at the beginning of the progression of temperature\nincrease (priming), determining its molecular interactions in detail is essential for developing strategies to\ncounteract global warming.\nThe manuscript is well written, the aim as well as the experimental procedures are clearly described. The conclusions and\nclaims are mostly supported by the data presented but can be strengthened by further evidence and a revised discussion.\nMajor criticisms and comments:\n1. Using exactly the same experimental approach presented in this manuscript (pull-down, Co-IP, ChIP, cycloheximide\ntreatment), Tan and colleagues (Tan et al., Sci. Adv. 9, 1738, 2023 “The heat response regulators HSFA1s promote\nArabidopsis thermomorphogenesis via stabilizing PIF4 during the day”) showed that in Arabidopsis plants treated at 28°C,\nHSFA1 interacted with, and stabilized PIF4 protein. Moreover, they showed that the interaction did not influence the PIF4\nDNA binding capacity. At the light of these previous results, which is the relationship between HSF1, PIF4, bZIPs? Do bZIPs\nbind PIF4 independently from HSF1? Or, do they form a ternary complex? How is the stability of PIF4 in the absence of both\nbZIPs and HSF1s?\nPlease, cite and discuss that paper in relationship to data presented here.\n2. Lines 158-160 and Figure 2C, top panels, BiFC experiments. Although GFP (and its half GFPN) is able to enter and exit\nthrough the nuclear pores, the most of it localizes in the cytosol. Therefore, the right negative control should be GFPN\ncontaining a nuclear localization signal (nGFPN). Morover, nGFPN + nGFPC (both with nls) should be also expressed\n3. Lines 175-179 and Figure 2D, E, F. Although in the absence of PIF4, the warmth-induced binding activity of different\nbZIPs on promoters of growth-promoting genes is reduced, it still remains significant compared to negative control, both at\n22°C and 29°C.\nCould it be an indication that, independently by PIF4 and temperature, bZIPs are able to promote transcription of PIF4 itself\nand growth-promoting genes?\n4. Figure 2B: the CoIP is performed by immunoselection bZIP17-HA and bZIP60-HA with anti-HA antibody, followed by blot\nincubation with anti-myc to detect PIF4-myc (IP, top and bottom panels). However, to assay the interaction of PIF4-myc with\nbZIP28-HA, PIF4-myc was first immunoselected with anti-myc, followed by blot with anti-HA. Since reciprocal CoIPs were\nnot done, do at least all the three IPs in the same manner, using the same antibody (anti-HA)\n5. Moreover, bZIP28N-HA seems to migrate too slow (63 kDa) compared with its expected molecular weight. Indeed, it\nmigrates around 48-50 kDa (see Suppl. Figure 1D). The HA epitope is 1-2 kDa. Please, check the positioning of MW marker.\n6. Figure 3E-F, graphics: although both graphs show a general susceptibility of PIF4-HA to degradation in the absence of\nbZIPs, the error bands are very wide and no statistical tests have been performed. Are these data really significant?\n7. Compared to the absence of bZIP17, the lack of bZIP28 has a greater effect on both PIF4 stability and hypocotyl\nelongation (Figure 3) and on PIF4 binding to promoters of growth-promoting genes (Figures 1 and 2D). Indeed,\noverexpression of bZIP28 stimulates hypocotyl growth more than the other two bZIPs (Figure 1F). The authors should\nhighlight and discuss this difference.\n8. what happens to the bZIPs/PIF4 complex if the temperature is raised? Does the bZIPs/PIF4 complex dissociate? What\nare the dynamics?\nMinor comments\n9. Methods: RNA expression\nActin is not a good housekeeping in treatment that promote hypocotyl elongation, because during growth, actin filaments are\nrearranged and increased. Ubiquitin could be better.\n10. Lines 42-43: the author state “whether a moderate temperature elevations induce ER stress remains unclear”. In a recent\npaper published on bioRvix “Dynamic regulation of protein homeostasis underlies acquired thermotolerance in Arabidopsis\nthaliana”\n(bioRxiv preprint doi: https://doi.org/10.1101/2023.08.04.552042), Bajaj et collegues show that\nUPR players bZIP60 or IRE1 are involved in priming-mediated heat stress recovery. Although the priming temperature is\nhigher than the 29°C used here, the study nevertheless highlights the involvement of the UPR in moderate\nheat stress as a protective mechanism on subsequent severe heat stress. Please, revise the statement and citate and discuss the Bajaj’s paper.", "reviewer_2": "Thermomorphogenesis refers to the plant's responses to a slight rise in temperature, encompassing changes in\ndevelopment, growth, metabolism, and immunity. The transcription factor PIF4 plays a central role in this process: warm\ntemperatures promote its expression, protein accumulation, and activity, which in turn activate genes involved in auxin\nbiosynthesis and signaling. However, how plants sense and transduce temperature signals at the cellular level to regulate\nPIF4 protein is still not fully understood. In this manuscript, the authors report a novel link between ER Unfolded Protein\nResponse (UPR) and thermomorphogenesis in plants. The authors showed that three UPR-associated bZIP transcription\nfactors, bZIP17, bZIP28, and bZIP60, can promote hypocotyl growth in a PIF4-dependent manner. Active ZIP factors form\ncomplexes with PIF4 in the nucleus, and PIF4 proteins are stabilized by the bZIP factors.\nOverall, this study attempted to reveal a novel signaling pathway in which warm temperatures stabilize PIF4 by activating\nER stress and the Unfolded Protein Response (UPR), thereby promoting thermomorphogenesis. This discovery could offer\nsignificant insights into the cellular processes underlying plant temperature sensing and response. However, the study, at\nthis stage, would still need additional evidence to support the main conclusion, which addresses two main questions: 1) how\nwarm temperatures trigger the ER UPR response and 2) how the ER UPR intersects with the known temperature signaling\npathway and converges on PIF4. Here are my comments:\n1. This study demonstrates that warm temperatures promote the expression of BiP1, a UPR marker gene, as shown in\nSupplemental Figure 1B, to support the claim that warm temperature triggers the Unfolded Protein Response. However,\nwhether warm temperatures promote the nuclear translocation of bZIP factors—bZIP17N, bZIP28N, and bZIP60s—remains to be\nfurther validated. As reported in previous studies, the N-terminal component of bZIP28 is rapidly relocated to the\nnucleus after ER stress (Liu JX et al. Plant Cell. 2007. doi:10.1105/tpc.106.050021), nuclear translocation of the\nbZIPs is a crucial prerequisite for these factors to bind and stabilize PIF4. However, there is a lack of\ndirect evidence to support this mechanism.\n2. The model of bZIP-mediated PIF4 stabilization is very interesting. The current data using PIF4 overexpression lines\nsupport the hypothesis (Figure 3D). However, the caveat is that PIF4-HA does not accumulate to a higher level under\nwarmer temperatures in this transgenic line. Although the level of PIF4-HA was reduced in bzip28/60, this happened at both\ntemperatures. Since PIF4 antibodies are commercially available, it would be more convincing to examine the level of\nendogenous PIF4 and its stability in the bZIP mutants.\n3. Warm-temperature-induced PIF4 accumulation occurs early during the transition to warm temperatures; it would be helpful\nto show whether UPR signaling occurs kinetically earlier than the PIF4 responses.\n4. In Figure 2, the authors used a transgenic line that overexpressed PIF4, bZIP17N, bZIP28N, and bZIP60s. It would be\ninformative to test whether the active bZIPs could enhance the function of PIF4 in this line.", "reviewer_4": "This manuscript presents a novel connection between the Unfolded Protein Response (UPR) and thermomorphogenesis in\nArabidopsis thaliana. The authors propose that the accumulation of the active (nuclear) forms of UPR-associated bZIP\ntranscription factors promotes hypocotyl elongation under warm temperatures by stabilizing and cooperating with the central\nthermomorphogenic regulator PIF4. Their findings suggest that UPR signaling, beyond its canonical stress response\nfunctions, contributes to the adaptive growth response known as thermomorphogenesis.\nWhile the conceptual link between UPR signaling and thermomorphogenesis is compelling and largely unexplored (despite\nknown crosstalk between UPR and factors such as HY5), the manuscript currently suffers from several significant issues.\nMechanistic insights remain superficial, and data interpretation frequently extends beyond what the results support.\nFurthermore, key experiments lack sufficient controls and quantitative rigor.\nSubstantial revisions are necessary to improve the statistical robustness, experimental design, and depth of mechanistic\ninterpretation. Overall i find this version very preliminary.\nMajor Comments:\nSupplemental Figure 2 – Tm Treatment: The reported effect of low-dose tunicamycin (Tm) on hypocotyl elongation is very\nweak. The presented images are not representative, and appear to be a compilation of different images, which should be\nexplicitly stated. Raw data and statistical analyses must be provided to support the conclusions. Without this, the\nphysiological relevance of Tm-induced elongation cannot be evaluated.\nLines 82–83 – Interpretation of Tm effects: The authors state that Tm promotes hypocotyl elongation in a PIF4-dependent\nmanner, but do not show whether low-dose Tm actually activates the UPR. This must be experimentally verified.\nThermomorphogenesis involves multiple traits, not just hypocotyl elongation. The manuscript should clarify whether other\nphenotypes were assessed (e.g., cotyledon expansion, leaf angle), and whether the effect is specific to hypocotyls or part of\na broader adaptive response.\nLines 84–87 – The rationale for using high-dose Tm (which inhibits growth) instead of the low-dose (which showed\nelongation) is unclear. The authors should explain why they shifted to a less physiologically relevant condition.\nLine 101 – UPR activity in bzip single mutants: It is unclear whether the bzip single mutants exhibit any UPR defects under\nwarmth. This is essential for understanding the individual contributions of bZIPs and the degree of redundancy.\nFigure 2C & Supplemental Figure 6 – Localization and BiFC controls: The nuclear localization of all proteins is expected,\nbut higher magnification images and quantitative colocalization analysis (e.g., Pearson correlation coefficients) are needed\nto support claims of interaction.\nWas the GFPN control fused to a nuclear localization signal (NLS)? If not, its absence from the nucleus does not serve as a\nvalid negative control.\nProtein–protein interactions: The interaction between bZIPs and PIF4 is central to the manuscript. However, no loss-of-\ninteraction mutants are used to validate the specificity of this interaction. Such mutants are essential as negative controls,\nespecially when overexpression lines dominate the study.\nLines 171–173 – ChIP-qPCR and target validation: Presumably, enrichment was assessed by qPCR, but this must be\nexplicitly stated. Negative control regions (e.g., distal promoter elements or coding regions) should be included. The use of\nPP2A as internal control is not clear. The authors should clarify whether PP2A is a PIF4 target or justify its selection. A\ngenome-wide ChIP-seq analysis would significantly strengthen the conclusions about co-occupancy and regulatory network\ndynamics.\nLine 179 – Chromatin accessibility hypothesis: The observed requirement for PIF4 in recruiting bZIPs could also reflect\nPIF4-induced chromatin remodeling, enhancing promoter accessibility. This alternative explanation should be discussed.\nFigure 3A–B – Missing controls: Wild-type controls are missing from these panels, limiting interpretability.\nFigure 3D – Blot quality and quantification: The immunoblots are overexposed and saturated, making them unsuitable for\nquantification. Quantification of band intensities lacks statistical analysis and error reporting. Results must be based on\nmultiple independent replicates.\nbZIP-dependent stability of PIF4: The stability of PIF4 is dependent on PHYB but also on posttranslational modifications.\nHow are bZIPs integrated in these mechanisms ? the authors do not touch at all this important aspect and therefore they\nmiss to provide an important link to the mechanism of PIF4 fate.\nLines 230–234: Thermomorphogenesis should be described as an adaptive response, not a developmental program. The\nclaim that UPR involvement in growth regulation is novel is incorrect—prior studies have demonstrated UPR involvement in\ndevelopmental processes, including root growth.\nDiscussion – Mechanistic context is missing. The discussion fails to integrate the findings into a broader\nmechanistic or physiological context.While the authors show that PIF4 is required for thermomorphogenesis and that bZIPs stabilize PIF4,\nthe biological relevance of this interaction remains vague. What advantage does this regulatory module confer? Under\nwhich physiological or stress scenarios is this pathway active?\nMinor Comments:\nLine 61: The activation of bZIP60 is mediated by IRE1-dependent unconventional splicing, not canonical pre-mRNA\nalternative splicing. This should be corrected.\nSupplemental Figure 1D: Clearly label that the blot refers to the active nuclear form of bZIP28. If possible, show the full-\nlength form in the same blot to distinguish activation from transcriptional induction.\nLine 98: Replace \"derived\" with more appropriate terms such as “UPR-associated” or “UPR-activated”.\nLine 107: Replace \"compromised\" with \"reduced\" to better reflect the observed changes.\nFigure 2A: Provide quantitative data from multiple replicates showing the percentage of bZIP pulled down relative to input.\nThe current figure lacks clarity and reproducibility."}, "author_response": "We thank the reviewer for the positive evaluation of our work and for recognizing that the manuscript\nis clearly written, with well-defined aims and procedures. We appreciate the suggestion to strengthen\nthe discussion and provide additional supporting evidence. In the revised manuscript, we have\nincorporated data from additional experiments and refined the discussion to better support our\nconclusions on the role of UPR-associated bZIPs in thermomorphogenesis.\nMajor criticisms and comments:\n1. Using exactly the same experimental approach presented in this manuscript (pull-down, Co-IP, ChIP,\ncycloheximide treatment), Tan and colleagues (Tan et al., Sci. Adv. 9, 1738, 2023 “The heat response\nregulators HSFA1s promote Arabidopsis thermomorphogenesis via stabilizing PIF4 during the day”)\nshowed that in Arabidopsis plants treated at 28°C, HSFA1 interacted with, and stabilized PIF4 protein.\nMoreover, they showed that the interaction did not influence the PIF4 DNA binding capacity. At the\nlight of these previous results, which is the relationship between HSF1, PIF4, bZIPs? Do bZIPs bind PIF4\nindependently from HSF1? Or, do they form a ternary complex? How is the stability of PIF4 in the\nabsence of both bZIPs and HSF1s? Please, cite and discuss that paper in relationship to data presented\nhere.\nWe thank the reviewer for pointing out the study by Tan et al. (Sci, Adv.2023), which provides\nimportant context for our work. We now cite this paper and discuss its main findings in the Result\nsection (lines 292-305) of our revised manuscript.\nOur data indicate that UPR-associated bZIPs stabilize PIF4 through a mechanism that is at least\npartially independent of HSFA1s. Specifically, pull-down assays revealed that bZIPs associate with\nHSFA1d, however such interactions are independent of PIF4 (Supplemental Figure 13A). Moreover, in\nthe hsfa1 quadruple mutant background, bZIPs still interact with PIF4, indicative of HSFA1-\nindependent binding. Collectively, these data suggest that bZIPs and HSFA1s likely act through\nseparate pathways to regulate PIF4 stabilization.\nBy interrogating the published RNA-seq data of short-term warm-treatment, we found an earlier\ninduction of bZIP28 and PIF4, compared to HSFA1a, further supporting the hypothesis that bZIPs\ncontribute to early stabilization of PIF4, while HSFA1s may reinforce PIF4 stability at later stages of\ncontinuing warmth. We have discussed this in the main text now (Line 298-305).\nRegarding PIF4 stability in the absence of both bZIPs and HSFA1s, while we completely agree that this\nserves as a very interesting research direction, combining such mutations can be extremely technically\nchallenging, given that hsfa1 quadruple already exhibits severe growth defects.\n2. Lines 158-160 and Figure 2C, top panels, BiFC experiments. Although GFP (and its half GFPN) is able\nto enter and exit through the nuclear pores, the most of it localizes in the cytosol. Therefore, the right\nnegative control should be GFPN containing a nuclear localization signal (nGFPN). Morover, nGFPN +\nnGFPC (both with nls) should be also expressed.\nWe thank the reviewer for the suggestion. GFPN naturally localizes to both the nucleus and the cytosol\nand is widely accepted as a standard negative control for BiFC and has been used in very recent studies\npublished in Nature Communications (Cai et al., 2025; Ge et al., 2025; Peng et al., 2025). More\nimportantly, the interaction between bZIPs and PIF4 has been further supported by diverse\ncomplementary approaches, including pull-down and Co-IP assays, confirming the results observed in\nBiFC assays.\n3. Lines 175-179 and Figure 2D, E, F. Although in the absence of PIF4, the warmth-induced binding\nactivity of different bZIPs on promoters of growth-promoting genes is reduced, it still remains\nsignificant compared to negative control, both at 22°C and 29°C. Could it be an indication that,\nindependently by PIF4 and temperature, bZIPs are able to promote transcription of PIF4 itself and\ngrowth-promoting genes?\nWe thank the reviewer for this comment. To further support the ChIP results, we have added EMSA\nassays showing that bZIP17N, bZIP28N, and bZIP60s can specifically bind to the G-box of PIF4 promoter\nin vitro (Figure 3D). Our ChIP assays (Figure 3E-G) show that these bZIPs are enriched at the promoters\nof PIF4, YUC8, and IAA19. While weak basal enrichment is detectable at 22°C, this is consistent with\nthe EMSA results and reflects minimal, non-temperature-induced binding. Promoter association is\nstrongly enhanced at 29°C, indicating that warmth specifically promotes bZIP binding. In the absence\nof PIF4, the warmth-induced occupancy is largely abolished, and only minimal residual binding\nremains. Consistently, overexpression of bZIPs does not rescue thermomorphogenic defects in pif4\nmutants. Together, these observations indicate that both promoter binding and functional activation\nof growth-promoting genes by bZIPs during thermomorphogenesis are strictly PIF4-dependent. We\nhave acknowledged this conclusion and included it in the revised manuscript (Lines 216-219).\n4. Figure 2B: the CoIP is performed by immunoselection bZIP17-HA and bZIP60-HA with anti-HA\nantibody, followed by blot incubation with anti-myc to detect PIF4-myc (IP, top and bottom panels).\nHowever, to assay the interaction of PIF4-myc with bZIP28-HA, PIF4-myc was first immunoselected\nwith anti-myc, followed by blot with anti-HA. Since reciprocal CoIPs were not done, do at least all the\nthree IPs in the same manner, using the same antibody (anti-HA).\nWe appreciate the reviewer’s constructive suggestion. In the revised manuscript, we have\nstandardized the Co-IP approach by using flag magnetic beads for immunoprecipitation, and detecting\nthe bait proteins with HA or myc antibody. Figure 3B has been updated accordingly. To further\nstrengthen these results, we performed reciprocal Co-IP experiments using PIF4-myc to\nimmunoprecipitated bZIP28N-HA and bZIP17N-HA (Supplemental Figure 8). Since we also generated\ndouble bZIP overexpression lines (Supplemental Figure 5C), distinct epitope tags were required;\naccordingly, we constructed bZIP60s-flag instead of bZIP60s-HA. These additional experiments\nconsistently support the interactions between PIF4 and the three bZIPs.\n5. Moreover, bZIP28N-HA seems to migrate too slow (63 kDa) compared with its expected molecular\nweight. Indeed, it migrates around 48-50 kDa (see Suppl. Figure 1D). The HA epitope is 1-2 kDa. Please,\ncheck the positioning of MW marker.\nWe thank the reviewer for raising this point. To clarify, we performed additional immunoblot analyses\nusing both a native bZIP28N antibody and an HA antibody (Figure R1). When seedlings were treated\nwith tunicamycin (Tm), a clear native bZIP28N band was detected around 48kDa in WT and pif4\nmutants but not in DMSO-treated seedlings or bzip28 mutant, confirming antibody specificity (Figure\nR1A and R1B). The same antibody also detected the bZIP28N-HA band in transgenic seedlings at an\napparent molecular mass of ~63kDa. Using an HA antibody, we further confirmed that bZIP28N-HA\nmigrates at an apparent size of ~63kDa, whereas the positive control PIF4-HA runs slightly above this\nposition (Figure R1C). Coomassie blue staining of the membrane verified equal sample loading (Figure\nR1D). Together, these results confirm that our molecular weight labeling is correct.\nWe should emphasize that migration position of a protein-SDS complex in an SDS gel is determined\nnot only by its molecular mass but also by several other factors including its shape (should be like a\nrigid rod) and its charged density. Anomalous migrations on SDS gels are not uncommon among\nproteins. Glycosylated proteins and histones are two examples that come to mind.\nFigure R1. Validation of bZIP28N-HA migration. (A) (B) Immunoblot with anti-bZIP28N antibody of WT,\npif4 treated with DMSO and Tm, bzip28 and bZIP28N-HA-OE, PIF4-HA-OE seedlings. (C) Immunoblot\nwith anti-HA detecting bZIP28N-HA and PIF4-HA seedlings. (D) Coomassie blue staining of the\nmembrane showing equivalent sample loading.\n6. Figure 3E-F, graphics: although both graphs show a general susceptibility of PIF4-HA to degradation\nin the absence of bZIPs, the error bands are very wide and no statistical tests have been performed.\nAre these data really significant?\nWe thank the reviewer for raising this point. In the revised manuscript, we have added statistical\nanalysis to Figure 4E and 4F. The results show that PIF4-HA protein degradation is significantly\naccelerated in the bZIP28/60 background compared to WT under warmth. The statistically significant\ndifferences are now indicated directly on the figures and figure legends. These results confirm that\nthe effect of bZIP28 and bZIP60 on PIF4 stability is robust and reproducible.\n7. Compared to the absence of bZIP17, the lack of bZIP28 has a greater effect on both PIF4 stability\nand hypocotyl elongation (Figure 3) and on PIF4 binding to promoters of growth-promoting genes\n(Figures 1 and 2D). Indeed, overexpression of bZIP28 stimulates hypocotyl growth more than the other\ntwo bZIPs (Figure 1F). The authors should highlight and discuss this difference.\nWe thank the reviewer for this suggestion. In the revised manuscript, we have highlighted and\ndiscussed different effects among bZIPs on PIF4 regulation (lines 329-346). In particular, bZIP28 and\nbZIP60 play dominant roles in thermomorphogenesis, consistent with their major functions in the\ncanonical UPR pathway, whereas the contribution of bZIP17 is rather modest. Seedlings\noverexpressing bZIP28N show longer hypocotyls under warm conditions, even at lower expression\nlevels as compared to bZIP17N-OE, while bZIP60s-OE lines display slightly reduced elongation, likely\ndue to their moderate UBQ-driven expression. bzip28/60 double mutants have shorter hypocotyls\nthan bzip17/60 mutants, and PIF4 stability assays confirm that bZIP28 and bZIP60 are more critical for\npromoting thermomorphogenic growth.\n8. what happens to the bZIPs/PIF4 complex if the temperature is raised? Does the bZIPs/PIF4 complex\ndissociate? What are the dynamics?\nWe thank the reviewer for this interesting question. We performed Co-IP assays in PIF4 and bZIP-\noverexpression lines at both 22°C and 29°C, and observed that the bZIP-PIF4 association is relatively\nstable at both temperatures, suggesting that once the complex is formed, it is maintained. The\ndetectable interaction at 22°C likely reflects the elevated protein levels in these overexpression lines.\nWe note that dynamic changes during early activation may not be fully captured in these assays. The\nshift of seedlings from 22°C to 29°C represents a dynamic change for UPR regulators, during which\nbZIPs translocate to the nucleus. These results are shown in in Figure 3B, Supplemental Figure 8 and\ndiscussed in the revised manuscript (lines 353-356).\nMinor comments\n9. Methods: RNA expression\nActin is not a good housekeeping in treatment that promote hypocotyl elongation, because during\ngrowth, actin filaments are rearranged and increased. Ubiquitin could be better.\nWe thank the reviewer for this comment. We agree that accurate normalization is particularly\nimportant for our time-course experiments, which capture the early transcriptional dynamics linking\nUPR activation with thermomorphogenesis. We repeated the time course qPCR assays using ubiquitin\nas a housekeeping control gene. As shown in Figure R2 (UBQ-normalized), bZIP28 and BiP1 are induced\nrapidly, within 20-30 min of warm treatment (R2A and R2B), whereas PIF4 and YUC8 show induction\nafter 40min (R2C and R2D). These induction patterns closely mirror those obtained with actin\nnormalization (Figure 1), demonstrating that the temporal sequence of early UPR gene activation\npreceding PIF4 induction is robust.\nTo further validate our previous observations, we also compared long-term warm-treatment\nexperiments (Figure R3, UBQ-normalized) with the actin-normalized data in Supplemental Figure 1. In\nboth cases, bZIP28 and BiP1 show similar induction dynamics, confirming that actin provides reliable\nnormalization in our analysis.\nImportantly, actin has been widely used as a qPCR reference in thermomorphogenesis studies (Lee et\nal., 2021; Tan et al., 2023; Fan et al., 2025). Together with our UBQ-based validation, these results\nsupport the robustness of our normalization strategy.\nFigure R2. Short-term warm treatment induces early UPR gene expression prior to PIF4 activation.\nqRT-PCR analysis of bZIP28 (A), BiP1(B), PIF4(C), and YUC8(D) transcript levels in seedlings shifted from\n22°C to 29°C. Expression was normalized to UBQ. Error bars represent the standard deviation (SD)\n(n=3).\nFigure R3. Long-term warm treatment shows consistent induction pattern of UPR and thermo-\nresponse genes. qRT-PCR analysis of bZIP28 (A), BiP1(B), PIF4(C), and YUC8(D) transcript levels in\nseedlings shifted from 22°C to 29°C. Expression was normalized to UBQ. Error bars represent the\nstandard deviation (SD) (n=3).\n10. Lines 42-43: the author state “whether a moderate temperature elevation induce ER stress\nremains unclear”. In a recent paper published on bioRvix “Dynamic regulation of protein homeostasis\nunderlies acquired thermotolerance in Arabidopsis thaliana” (bioRxiv preprint doi:\nhttps://doi.org/10.1101/2023.08.04.552042), Bajaj et collegues show that UPR players bZIP60 or IRE1\nare involved in priming-mediated heat stress recovery. Although the priming temperature is higher\nthan the 29°C used here, the study nevertheless highlights the involvement of the UPR in moderate\nheat stress as a protective mechanism on subsequent severe heat stress. Please, revise the statement\nand citate and discuss the Bajaj’s paper.\nWe thank the reviewer for drawing our attention to the work by Bajaj et al. (2023). In the revised\nmanuscript, we have clarified our statement on lines 43-45 to avoid the implication that no link\nbetween moderate heat and UPR has been reported. As suggested, we have also cited the study,\nwhich demonstrates the involvement of UPR components in priming-mediated thermotolerance at\ntemperatures higher than those tested here.\nReviewer #2 (Remarks to the Author):\nThermomorphogenesis refers to the plant's responses to a slight rise in temperature, encompassing\nchanges in development, growth, metabolism, and immunity. The transcription factor PIF4 plays a\ncentral role in this process: warm temperatures promote its expression, protein accumulation, and\nactivity, which in turn activate genes involved in auxin biosynthesis and signaling. However, how plants\nsense and transduce temperature signals at the cellular level to regulate PIF4 protein is still not fully\nunderstood. In this manuscript, the authors report a novel link between ER Unfolded Protein Response\n(UPR) and thermomorphogenesis in plants. The authors showed that three UPR-associated bZIP\ntranscription factors, bZIP17, bZIP28, and bZIP60, can promote hypocotyl growth in a PIF4-dependent\nmanner. Active ZIP factors form complexes with PIF4 in the nucleus, and PIF4 proteins are stabilized\nby the bZIP factors.\nOverall, this study attempted to reveal a novel signaling pathway in which warm temperatures\nstabilize PIF4 by activating ER stress and the Unfolded Protein Response (UPR), thereby promoting\nthermomorphogenesis. This discovery could offer significant insights into the cellular processes\nunderlying plant temperature sensing and response. However, the study, at this stage, would still need\nadditional evidence to support the main conclusion, which addresses two main questions: 1) how\nwarm temperatures trigger the ER UPR response and 2) how the ER UPR intersects with the known\ntemperature signaling pathway and converges on PIF4. Here are my comments:\nWe thank the reviewer for the nice summary of our study. We appreciate the reviewer’s recognition\nof the novel connection between the UPR and thermomorphogenesis, highlighting the role of bZIP17,\nbZIP28, and bZIP60 in stabilizing PIF4 and promoting hypocotyl growth under the warmth.\nWe also value the reviewer’s emphasis on the two central questions regarding (1) how moderate\nwarm temperatures activate the UPR and (2) how the UPR intersects with established temperature\nsignaling to regulate PIF4. In the revised manuscript, we have addressed these points by providing\nadditional data and discussion clarifying the early activation of UPR components under warm\nconditions, the PIF4 dependence of bZIP-mediated transcriptional regulation, and the functional\ninterplay between bZIPs and PIF4 in thermomorphogenesis.\nThese clarifications have been further discussed in the revised manuscript to strengthen the link\nbetween UPR activation and PIF4-dependent thermormophogenic growth.\n1. This study demonstrates that warm temperatures promote the expression of BiP1, a UPR marker\ngene, as shown in Supplemental Figure 1B, to support the claim that warm temperature triggers the\nUnfolded Protein Response. However, whether warm temperatures promote the nuclear\ntranslocation of bZIP factors—bZIP17N, bZIP28N, and bZIP60s—remains to be further validated. As\nreported in previous studies, the N-terminal component of bZIP28 is rapidly relocated to the nucleus\nafter ER stress (Liu JX et al. Plant Cell. 2007. doi:10.1105/tpc.106.050021), nuclear translocation of the\nbZIPs is a crucial prerequisite for these factors to bind and stabilize PIF4. However, there is a lack of\ndirect evidence to support this mechanism.\nWe thank the reviewer for this comment. The nuclear accumulation of bZIP factors under warm\nconditions has indeed been addressed in our study. As shown in Supplemental Figure 1D, bZIP28N is\ndetectable in seedlings after 2 days of warm treatment, with Tm-treated WT included as a positive\ncontrol for bZIP28N production. These results indicate that bZIP28N is processed and available to\ntranslocate to the nucleus under warm conditions, supporting its stability to bind and stabilize PIF4.\n2. The model of bZIP-mediated PIF4 stabilization is very interesting. The current data using PIF4\noverexpression lines support the hypothesis (Figure 3D). However, the caveat is that PIF4-HA does not\naccumulate to a higher level under warmer temperatures in this transgenic line. Although the level of\nPIF4-HA was reduced in bzip28/60, this happened at both temperatures. Since PIF4 antibodies are\ncomercially available, it would be more convincing to examine the level of endogenous PIF4 and its\nstability in the bZIP mutants.\nWe thank the reviewer for this valuable comment. In the revised manuscript, we have re-labelled the\nquantified protein band intensities in Figure 4D, wherein the values represent the ratio of target\nprotein to actin loading intensity. The updated labeling clearly shows that PIF4-HA accumulates to\nhigher levels under warm conditions. In addition, our original submission already included the analysis\nof endogenous PIF4 protein levels in both mutant and overexpression lines (Supplemental Figure 9;\nLines 231-237), which further supports our conclusion.\n3. Warm-temperature-induced PIF4 accumulation occurs early during the transition to warm\ntemperatures; it would be helpful to show whether UPR signaling occurs kinetically earlier than the\nPIF4 responses.\nWe thank the reviewer for this insightful comment. To address this question, we performed a time\ncourse assay with short-term thermo treatment to monitor the temporal dynamics of UPR activation\nrelative to PIF4 induction (Figure 1, lines 59-63). These experiments show that UPR signaling, indicated\nby early induction of bZIP28 and BiP1, occurs prior to PIF4 transcript accumulation and activation of\nits target gene YUC8, demonstrating that UPR components are engaged earlier than canonical\nthermoresponsive genes during the transition to warm temperatures.\n4. In Figure 2, the authors used a transgenic line that overexpressed PIF4, bZIP17N, bZIP28N, and\nbZIP60s. It would be informative to test whether the active bZIPs could enhance the function of PIF4\nin this line.\nWe thank the reviewer for the suggestion. In our experiments, PIF4 overexpression lines already\nexhibit maximal hypocotyl elongation under warm conditions. Consequently, introducing active bZIPs\ninto this background does not further enhance elongation, as the phenotype might be already\nsaturated. Thus, we believe that our current data sufficiently demonstrate that bZIPs promote\nthermomorphogenesis in a PIF4-dependent manner.\nReviewer #4 (Remarks to the Author):\nThis manuscript presents a novel connection between the Unfolded Protein Response (UPR) and\nthermomorphogenesis in Arabidopsis thaliana. The authors propose that the accumulation of the\nactive (nuclear) forms of UPR-associated bZIP transcription factors promotes hypocotyl elongation\nunder warm temperatures by stabilizing and cooperating with the central thermomorphogenic\nregulator PIF4. Their findings suggest that UPR signaling, beyond its canonical stress response\nfunctions, contributes to the adaptive growth response known as thermomorphogenesis.\nWhile the conceptual link between UPR signaling and thermomorphogenesis is compelling and largely\nunexplored (despite known crosstalk between UPR and factors such as HY5), the manuscript currently\nsuffers from several significant issues. Mechanistic insights remain superficial, and data interpretation\nfrequently extends beyond what the results support. Furthermore, key experiments lack sufficient\ncontrols and quantitative rigor.\nSubstantial revisions are necessary to improve the statistical robustness, experimental design, and\ndepth of mechanistic interpretation. Overall I find this version very preliminary.\nWe thank the reviewer for this thoughtful summary as well as constructive evaluation and suggestions.\nIn the revised version, we have included new data that adds new molecular evidence on how UPR\nregulates thermomorphogenesis, repeated key experiments with additional controls and expanded\nthe discussion section, in particular of newly added results.\nMajor Comments:\nSupplemental Figure 2 – Tm Treatment: The reported effect of low-dose tunicamycin (Tm) on\nhypocotyl elongation is very weak. The presented images are not representative, and appear to be a\ncompilation of different images, which should be explicitly stated. Raw data and statistical analyses\nmust be provided to support the conclusions. Without this, the physiological relevance of Tm-induced\nelongation cannot be evaluated.\nWe thank the reviewer for pointing out this issue. In the original version, while the images were taken\nfrom the same experimental plate, corresponding plants were placed on the opposite edges, which\nresulted in a slightly different background reflection. We apologize for such confusion and to avoid\nfurther misinterpretation, we have revised the figure by selecting uniformly bright, representative\nseedlings from the same plate and replacing the earlier image. We also provide the full raw\nmeasurements of hypocotyl lengths under Tm treatment in the Supplemental Source Data and include\nthe corresponding statistical analyses in the updated Supplemental Figure 2. While the elongation\nresponse to low-dose Tm is modest, it is consistent and statistically significant across independent\nreplicates. We have therefore refined our description in the Results to present this effect as a partial\nmimic of warm-induced elongation rather than a strong induction.\nLines 82–83 – Interpretation of Tm effects: The authors state that Tm promotes hypocotyl elongation\nin a PIF4-dependent manner, but do not show whether low-dose Tm actually activates the UPR. This\nmust be experimentally verified. Thermomorphogenesis involves multiple traits, not just hypocotyl\nelongation. The manuscript should clarify whether other phenotypes were assessed (e.g., cotyledon\nexpansion, leaf angle), and whether the effect is specific to hypocotyls or part of a broader adaptive\nresponse.\nWe appreciate this valuable comment. To verify whether Tm activates the UPR at low dose, we\nexamined the expression of UPR marker genes upon low-dose Tm treatment. As shown in\nSupplemental Figure 2D and 2E, mild Tm treatment led to significantly enhanced transcript abundance\nof both BiP1 and bZIP28, indicative of UPR activation. Moreover, transcript accumulation of both\ngenes was further enhanced at 29 °C, consistent with our conclusion that elevated temperature adds\non ER stress. These results together show that mild Tm treatment is sufficient to induces UPR signaling\neffectively.\nRegarding thermomorphogenesis traits, in this study we focused on hypocotyl elongation, which is\nthe most prominent and widely used marker in seedlings. We have clarified this emphasis and further\ndiscussed in the revised manuscript (lines 88-90).\nLines 84–87 – The rationale for using high-dose Tm (which inhibits growth) instead of the low-dose\n(which showed elongation) is unclear. The authors should explain why they shifted to a less\nphysiologically relevant condition.\nWe thank the reviewer for this observation. In the original manuscript, we noted that the Tm\nconcentration used for ER stress analysis caused growth defects, which may not have been sufficiently\nemphasized. To make this clearer, we have revised the text to explain the rationale more explicitly.\nSpecifically, we now state that activation of UPR signaling by pharmacological agents such as Tm and\nDTT typically results in strong seedling growth arrest and chlorosis, likely due to drug overdose, which\nis in contrast with the growth-promoting effects observed during thermomorphogenesis (lines 85-88).\nMoreover, we agree with the reviewer that such contrasting effects of different Tm concentrations on\nplant growth serve as an interesting future research direction, which is, however beyond the scope of\nthe present study.\nLine 101 – UPR activity in bzip single mutants: It is unclear whether the bzip single mutants exhibit any\nUPR defects under warmth. This is essential for understanding the individual contributions of bZIPs\nand the degree of redundancy.\nWe appreciate the reviewer’s comment. We observed that single mutants of bzip17, bzip28, or bzip60\ndo not display detectable hypocotyl defects under warm conditions, consistent with their minimal\ngrowth phenotypes in response to Tm treatment. These observations, together with previous reports\n(Kim et al., 2018; Kim et al., 2022), support the conclusion that these bZIPs function redundantly in\nregulating UPR signaling and thermomorphogenesis. To improve clarity, we have highlighted this point\nmore explicitly in the Discussion (Lines 343-346). Further dissection of distinct roles of individual bZIPs\nin both UPR and thermomorphogenesis would be interesting and particularly informative on UPR\nsignaling. Such quantitative analysis however requires genetic and molecular assays at a very high\nresolution, which are unfortunately extremely technical challenging with current prevailing\nmethodology. Given the robust phenotypes observed with both loss- and gain-of-function bZIP\nmutants we analyzed, we believe these are sufficient to claim the major role of UPR signaling in\nregulating thermomorphogenesis.\nFigure 2C & Supplemental Figure 6 – Localization and BiFC controls: The nuclear localization of all\nproteins is expected, but higher magnification images and quantitative colocalization analysis (e.g.,\nPearson correlation coefficients) are needed to support claims of interaction.\nWe appreciate the reviewer’s suggestion. In the revised manuscript, we have replaced the\ncolocalization images with higher magnification versions to better illustrate the nuclear overlap\namong candidate proteins (Supplemental Figure 7A). We also performed PCC analysis with a score of\napproximately 0.5, indicative of nuclear co-localization between active forms of bZIPs and PIF4\n(Supplemental Figure 7B; lines 234-237). These additions reinforce the evidence for in vivo protein-\nprotein interactions.\nWas the GFPN control fused to a nuclear localization signal (NLS)? If not, its absence from the nucleus\ndoes not serve as a valid negative control.\nWe thank the reviewer for the suggestion. GFPN naturally localizes to both the nucleus and the cytosol\nand is widely accepted as a standard negative control for BiFC and has been used in very recent studies\npublished in Nature Communications (Cai et al., 2025; Ge et al., 2025; Peng et al., 2025). More\nimportantly, the interaction between bZIPs and PIF4 has been further supported by diverse\ncomplementary approaches, including pull-down and Co-IP assays, confirming the results observed in\nBiFC assays.\nProtein–protein interactions: The interaction between bZIPs and PIF4 is central to the manuscript.\nHowever, no loss-of-interaction mutants are used to validate the specificity of this interaction. Such\nmutants are essential as negative controls, especially when overexpression lines dominate the study.\nWe thank the reviewer for this important comment. Identifying mutant variants with defects in\nassociation with specific interacting partners requires intensive and comprehensive biochemical\nanalysis in couple with genetic validation, which often needs years of dedicative works. While we\nwould appreciate the exist of such mutant variants that can serve as an additional layer of negative\ncontrol, we believe our current methodology in analyzing protein-protein interaction is technically\nvalid and widely accepted in similar studies, which includes multipronged, complementary methods\nwith relevant controls. Together, these complementary lines of evidence robustly support the\nspecificity of the PIF4-bZIP interaction.\nLines 171–173 – ChIP-qPCR and target validation: Presumably, enrichment was assessed by qPCR, but\nthis must be explicitly stated. Negative control regions (e.g., distal promoter elements or coding\nregions) should be included. The use of PP2A as internal control is not clear. The authors should clarify\nwhether PP2A is a PIF4 target or justify its selection. A genome-wide ChIP-seq analysis would\nsignificantly strengthen the conclusions about co-occupancy and regulatory network dynamics.\nWe thank the reviewer for these suggestions. We have clarified in the revised manuscript that\nenrichment in our ChIP experiments was assessed by qPCR. The qPCR primers used to detect target\ngene promoters were selected based on previously published studies (Yang et al., 2021; Tan et al.,\n2023), ensuring reliable detection of PIF4 binding regions. PP2A was used as a negative control, as in\nprior publications (Maeda et al., 2018; Yang et al., 2021; Huang et al., 2024), because it is not a known\nPIF4 target and provides a baseline for non-specific enrichment. While we agree that genome-wide\nChIP-seq would provide a more comprehensive view of co-occupancy and regulatory network\ndynamics, this is beyond the scope of the current study, and we aim to address such questions in\nfuture study. Nevertheless, the targeted ChIP-qPCR experiments, together with our biochemical and\nimaging evidence, provide robust support for PIF4 interaction with active bZIPs at specific loci.\nLine 179 – Chromatin accessibility hypothesis: The observed requirement for PIF4 in recruiting bZIPs\ncould also reflect PIF4-induced chromatin remodeling, enhancing promoter accessibility. This\nalternative explanation should be discussed.\nWe thank the reviewer for this important suggestion. We have addressed this point in the revised\nDiscussion (lines 326-328), acknowledging that while our data indicate that active bZIPs can directly\nbind target promoters, we cannot exclude the possibility that PIF4 additionally facilitates bZIPs\nrecruitment by enhancing chromatin accessibility. To further support direct DNA binding, we have\nadded EMSA analysis showing that the active bZIPs can bind to the G-box motif in vitro (Figure 4D).\nTogether, these results suggest that direct promoter binding by bZIPs might function as the key\nmechanism, while also noting the potential contribution of PIF4-mediated chromatin remodeling.\nFigure 3A–B – Missing controls: Wild-type controls are missing from these panels, limiting\ninterpretability.\nWe thank the reviewer for the comment. In these experiments, the primary comparison is between\nPIF4-HA overexpression in the WT background (PIF4-OE/WT) and in bzip double mutants (PIF4-\nOE/bzip17/60 and PIF4-OE/bzip28/60). The PIF4-OE/WT, therefore, serves as a more biologically\nrelevant control for assessing the effect of bZIP loss on PIF4-dependent phenotype in a comparative\nmanner. Nevertheless, to improve clarity and interpretability, we have now included WT samples in\nthe updated Figure 4A and 4B.\nFigure 3D – Blot quality and quantification: The immunoblots are overexposed and saturated, making\nthem unsuitable for quantification. Quantification of band intensities lacks statistical analysis and\nerror reporting. Results must be based on multiple independent replicates.\nWe thank the reviewer for this comment. The experiment was repeated with 3 independent biological\nsamples, and similar patterns were consistently observed. The corresponding source data are\nprovided in the Supplemental Source Data.\nbZIP-dependent stability of PIF4: The stability of PIF4 is dependent on PHYB but also on\nposttranslational modifications. How are bZIPs integrated in these mechanisms? the authors do not\ntouch at all this important aspect and therefore they miss to provide an important link to the\nmechanism of PIF4 fate.\nWe thank the reviewer for raising this important point. We have now addressed the interplay between\nbZIPs and the PIF4-phyB module in the revised manuscript (lines 263-291). Our data show that\noverexpression of bZIP28N or bZIP60s partially restores PIF4 accumulation and thermomorphogenic\nresponses in 35S::YHB plants, which express a constitutively active phyB that strongly suppresses PIF4\nfunction. We further explored underlying molecular mechanisms by using a combination of\nbiochemical assays. In particular, pull-down and BiFC assays demonstrate that bZIP17N, bZIP28N, and\nbZIP60s directly interact with phyB, and LCI assays indicate that bZIP28N and bZIP60s attenuate the\nPIF4-phyB interaction in vivo. Together, these results suggest that bZIPs stabilize PIF4 by partially\ncounteracting phyB-mediated inhibition, providing a mechanistic link between UPR signaling and PIF4\nfate under warm conditions.\nLines 230–234: Thermomorphogenesis should be described as an adaptive response, not a\ndevelopmental program. The claim that UPR involvement in growth regulation is novel is incorrect—\nprior studies have demonstrated UPR involvement in developmental processes, including root growth.\nWe thank the reviewer for this comment. The manuscript has been revised to describe\nthermomorphogenesis as an adaptive response and to adjust the wording regarding UPR involvement\nin growth regulation.\nDiscussion – Mechanistic context is missing. The discussion fails to integrate the findings into a broader\nmechanistic or physiological context. While the authors show that PIF4 is required for\nthermomorphogenesis and that bZIPs stabilize PIF4, the biological relevance of this interaction\nremains vague. What advantage does this regulatory module confer? Under which physiological or\nstress scenarios is this pathway active?\nWe thank the reviewer for this comment. We have revised the Discussion to better integrate our\nfindings into a broader physiological context. The manuscript now highlights that the interaction\nbetween bZIPs and PIF4 provides a mechanism for coordinating UPR signaling with\nthermomorphogenic growth, ensuring adaptive hypocotyl elongation under warm conditions. This\nregulatory module allows seedlings to fine-tune growth in response to mild ER stress that can be\ninduced by elevated temperatures, linking environmental cues to cellular stress responses.\nMinor Comments:\nLine 61: The activation of bZIP60 is mediated by IRE1-dependent unconventional splicing, not\ncanonical pre-mRNA alternative splicing. This should be corrected.\nWe thank the reviewer for this comment. The manuscript has been revised to correctly describe\nbZIP60 activation as mediated by IRE1-dependent unconventional splicing.\nSupplemental Figure 1D: Clearly label that the blot refers to the active nuclear form of bZIP28. If\npossible, show the full-length form in the same blot to distinguish activation from transcriptional\ninduction.\nWe thank the reviewer for this comment. The antibody used for bZIP28 was generated in rabbit\nagainst bZIP28N, and even after purification it produces multiple unspecific bands, given its polyclonal\nantibody nature. Because warm-induced ER stress causes only mild accumulation of the active nuclear\nform, longer exposure is required to visualize the bZIP28N band. To clarify the specificity of the signal,\nwe provide a new figure (Figure R1) showing the longer exposure along with a Tm-treated sample to\nillustrate the pattern of unspecific bands. The active nuclear form is clearly labeled in this figure.\nLine 98: Replace \"derived\" with more appropriate terms such as “UPR-associated” or “UPR-activated”.\nWe thank the reviewer for this comment. The manuscript has been revised to replace “derived” with\n“UPR-associated”.\nLine 107: Replace \"compromised\" with \"reduced\" to better reflect the observed changes.\nWe thank the reviewer for this comment. The manuscript has been revised to replace “compromised”\nwith “reduced”.\nFigure 2A: Provide quantitative data from multiple replicates showing the percentage of bZIP pulled\ndown relative to input. The current figure lacks clarity and reproducibility.\nWe thank the reviewer for this comment. We have updated Figure 3A with a revised pull-down image\nto improve clarity. Consistent with published studies using similar assays, we did not include a\nquantitative chart because the pull-down primarily serves as a qualitative demonstration of protein-\nprotein interaction. Importantly, the interaction was reproducibly observed across independent\nexperiments."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In this revised version of the manuscript NCOMMS-25-41112A - \"Unfolded protein response bZIP factors interact with PIF4 to promote thermomorphogenesis\" by Chua et collegues, the authors have answered to the criticisms by discussing the relevant issues raised. The inclusion and reorganization of new experimental data and additional controls, and the revision of the discussion, strengthen and clarify the conclusions.", "reviewer_2": "The authors have performed new experiments to support their conclusions and have also adequately addressed all of my questions.", "reviewer_4": "The new version of the manuscript has addressed some of my comments with additional data or clarifications. One comment that was not addressed is the qPCR-ChIP, where the authors have amplified only the promoter region of interest (which is fine) but i believe that other distal regions should be also shown as negative controls. This is typical for ChIP."}, "author_response": "We thank the reviewer for the positive evaluation of our revisions. We are pleased that the additional\nexperiments, newly incorporated controls, and the reorganization of the manuscript have clarified our\nconclusions and addressed the issues raised in the previous review. We appreciate the reviewer’s\nconstructive feedback, which has significantly improved the quality and clarity of the manuscript.\n\nWe thank the reviewer for the positive assessment. We are pleased that the additional experiments\nand our revisions have satisfactorily addressed all previous concerns. We appreciate the reviewer’s\nconstructive feedback, which has helped improve the clarity and strength of the manuscript.\n\nWe thank the reviewer for this helpful suggestion. In our ChIP-qPCR-ChIP analysis, binding specificity is\ndemonstrated through the use of PP2A as an internal negative control locus, which does not contain\nthe G-box motif. Additionally, using the same promoter amplicon, we observe strong and reproducible\ntemperature-dependent differences in enrichment (22 °C and 29 °C), indicating that the ChIP signal\nreflects regulated and sequence-specific binding rather than non-specific background.\nSeveral recent Nature Communications papers using ChIP-qPCR similarly rely on a single promoter\nregion with an internal negative control locus, without an additional distal amplicon, and our approach\nfollows this established practice (Cheng et al., 2024; Guo et al., 2025; Wang et al., 2025)."}]

Decision letter