Oligomerization-competent PIF4 drives thermomorphogenesis through functional redundancy in transactivation and DNA binding

Domain: biology
Published: 2026-03-17
Editorial outcome: major_revision

Abstract
Plants tailor their architecture to warm temperatures through the central transcription factor
PHYTOCHROME-INTERACTING FACTOR 4 (PIF4). Here, we dissect how structured and
disordered regions of PIF4 contribute to its function in thermomorphogenesis. A long N-terminal
intrinsically disordered region (IDR) enables PIF4 to form low-mobility condensates. Within this IDR,
we identify an acidic transactivation domain (TAD) and an extended basic segment that carries a
nuclear-localization signal and the canonical basic motif of the basic helix-loop-helix (bHLH) domain.
The basic segment is both necessary and sufficient to drive PIF4 condensate formation, while the TAD
tunes condensate properties. Strikingly, alanine substitutions that abolish TAD-mediated transactivation,
disrupt DNA binding, or greatly reduce phase-separation propensity have no significant effect on
thermomorphogenetic hypocotyl elongation. By contrast, substituting twelve basic residues within the
basic segment, which disrupts both DNA binding and HLH-mediated oligomerization, abolishes
thermo-induced hypocotyl growth. These findings suggest that PIF4’s oligomerization competence
contributes significantly to thermomorphogenesis by enabling partner recruitment, allowing DNA-
binding and transactivation functions to be supplied in trans.

Introduction
Plants react distinctly to different temperature regimes. Growth and development are severely impeded
at extreme heat or cold, as cell damage hinders normal physiological processes and energy and
resources are redirected from growth to stress tolerance and defense1,2. By contrast, within the non-
stressful ambient range (≈12–27 °C), moderate warming strongly remodels seedling morphology—
hypocotyls, roots, and petioles elongate, reshaping overall architecture in a process known as
thermomorphogenesis3,4. These changes are driven by multiple thermosensory transcription factors
(TTFs), which accumulate and/or activate as temperature rises and regulate genes involved in
phytohormone signaling and cell-wall modifications5,6.

Arabidopsis thaliana TTFs identified to date span multiple gene families, including but not limited to
PHYTOCHROME-INTERACTING FACTORs (PIFs), B-BOX proteins (BBXs), Class A1 HEAT
SHOCK FACTORs (HSFA1s), TEOSINTE BRANCHED 1/CYCLOSIDEA/PCF proteins (TCPs), and
BRASSINAZOLE RESISTANT 1 (BZR1)7–17. At the hub of their regulatory network is the basic Helix-
Loop-Helix (bHLH) transcription factor PIF4, as nearly every other TTF converges on its pathway by
modulating at least one aspect of PIF4—its transcription, DNA-binding, transcriptional activity, and
protein stability18. For example, BZR1 and TCP5 bind the PIF4 promoter and boost its expression upon
temperature elevation12,13,17, whereas thermo-induced nuclear accumulation of HSFA1s not only
enhances PIF4 transcription but also stabilizes PIF4 protein15,16. PIF4’s pivotal status in thermosensory
growth is underscored by its position immediately downstream of the two established thermosensors—
phytochrome B (phyB) and the evening-complex component ELF319–24. Multiple BBX proteins,
including BBX18, BBX23, BBX24, and BBX25, have been reported to promote PIF4-driven
thermomorphogenesis by physically interacting with and antagonizing ELF39–11.

PIF4’s centrality is unsurprising given that the eight-member PIF family (PIF1–PIF8) underpins
numerous pathways, including photomorphogenesis, shade avoidance, temperature signaling, and
immunity25–27. Acting as a signaling hub, PIF4 integrates diverse cues—light, temperature, drought,
salinity, and pathogen attack—and thereby mediates growth-defense trade-offs, a role exemplified by its
control of leaf senescence28–36. Yet, aside from two sequence features shared by all PIFs—an N-terminal
active phyB-binding (APB) motif that confers phyB interaction and a C-terminal bHLH domain for
DNA binding and oligomerization37, the sequence-function landscape of PIF4 remains largely
unexplored.

Eukaryotic transcription factors (TFs) are characterized by two core functions: a DNA-binding capacity
that recognizes specific sequences (e.g., cis-elements in promoters and enhancers) and a transactivation
activity that recruits cofactors to modulate transcription38,39. Accordingly, TFs are generally organized
into a DNA-binding domain (DBD) and one or more effector domains (EDs), such as transcriptional
activation domains (TADs) and repression domains (RDs)40–43. Many TFs also include auxiliary regions
that mediate protein-protein interactions (e.g., oligomerization) and other regulatory processes (e.g.,
post-translational modifications). These contacts with DNA or protein cofactors arise from both ordered
domains, such as DBDs or oligomerization domains, and intrinsically disordered regions (IDRs) that
can adopt multiple conformations44. Mounting evidence shows that multivalent interactions among
IDRs drive phase separation to form biomolecular condensates that facilitate transcription45. Notably,
many TADs partition with the Mediator coactivator complex into such condensates, linking a TF’s
activation potential to the phase-separation propensity of its TAD46. Though some DBDs (e.g., zinc
finger and homeodomain) allow TFs to bind DNA as monomers, many TFs engage DNA as dimers or
higher-order oligomers47. This requirement is especially pronounced in the bHLH, bZIP, and MADS-
box families, whose noncovalent dimerization or oligomerization is a prerequisite for DNA binding.
Emerging evidence suggests that dimerization/oligomerization also contributes to the nuclear
condensate formation of several TFs48,49.

PIF4 is a typical bHLH TF. It carries a bipartite DBD: a basic stretch rich in arginines that contacts
DNA and an HLH module that drives both homo- and hetero-oligomerization50–52. Upon warm-
temperature cues, PIF4 accumulates in the nucleus and binds promoters of growth-related genes—
particularly those governing auxin biosynthesis and signaling, such as YUC8, IAA19, and IAA297,53,54.
PIF4 also physically engages several Mediator subunits, including MED14 and MED25, to activate
thermo-induced gene expression55,56, implying the existence of a functional TAD. In PIF3, a close
homolog, the transactivation activity maps to a p53-like TAD featured by a ΦxxΦΦ motif (Φ,
hydrophobic residues)57. Mutating this motif dramatically disrupts PIF3’s transcriptional activation
activity both in yeast and in planta, whereas an equivalent mutation in PIF4 only modestly reduces its
activity, leaving its precise TAD undefined. Recent work shows that PIF4 can phase separate with
JASMONATE ZIM-domain protein 3 (JAZ3), which restricts PIF4’s DNA-binding and transcriptional
activation activities58. However, whether PIF4 phase separation is indeed crucial for its
thermomorphogenetic function and whether the condensate formation relies on an as-yet-unmapped
TAD, its oligomeric bHLH core, or both remains unknown.

In this study, we dissect whether and how PIF4’s structured bHLH core and its “dark-matter” IDR
contribute to plant thermomorphogenesis. We uncover a long N-terminal IDR that forms low-mobility
condensates in vitro and in planta, yet these assemblies are temperature-insensitive and exhibit limited
fluidity. Interestingly, eliminating phase separation—by either mutating the acidic-type TAD or
substituting basic residues in the DNA-binding motif—barely impairs PIF4’s ability to restore warm
temperature-induced hypocotyl elongation in the pif4 mutant. By contrast, substituting twelve basic
residues, nine in the basic motif and three in the first helix of PIF4’s HLH domain, abolishes higher-
order oligomerization and extinguishes thermomorphogenetic growth. We therefore propose a partner-
dependent model of PIF4-mediated thermomorphogenetic regulation, in which PIF4’s ability to
oligomerize—potentially aided by its long N-terminal IDR—enables recruitment of cofactors that can
supply DNA-binding and transactivation in trans.

Results
PIF4 forms low-mobility condensates that are insensitive to ambient temperatures
PIF4 was reported to form nuclear speckles in plants58,59. Some key thermosensory transcription factors,
such as HSFA1a, undergo phase transition to liquid droplets as a function of temperature60. To
investigate whether the formation of PIF4 nuclear speckles is affected by ambient temperatures, we
expressed mGFPmut2-tagged PIF4 in Nicotiana benthamiana leaves through Agro-infiltration and
compared nuclear speckle formation under cooler (20 °C) and warmer temperatures (27 °C). At both
temperatures, mGFPmut2-PIF4 proteins were localized to distinct nuclear speckles and nucleoplasm
(Fig. 1a). The number and size of these granules were comparable between 20 °C and 27 °C (Fig. 1b–d).
We further applied fluorescence recovery after photobleaching (FRAP) to compare the liquidity of
mGFPmut2-PIF4 nuclear speckles in tobacco leaves. The fluorescent signal in the photobleached region
marginally recovered at both temperatures (Fig. 1e–g), suggesting an ambient temperature-insensitive,
low-mobility structure of mGFPmut2-PIF4 condensates.

Unlike HSFA1a, ELF3, and PIF724,59,61, which possess prion-like low complexity domains (PrLDs),
PIF4 does not display a high-confidence PrLD score in PLAAC58. We hypothesize that PIF4 harbors
other IDRs that may contribute to its phase separation in plants. Indeed, IUPred362 predicts a long IDR
in the N-terminus of PIF4 protein (Fig. 1h). This PIF4IDR, spanning from 30 to 288 amino acids,
includes a long unstructured region (57-219 a.a.) and also some known domains/motifs, such as most of
the Active Phytochrome B-binding (APB) domain, the nuclear localization signal (NLS), the basic
DNA-binding region, and the first helix in the HLH dimerization/tetramerization domain51. We fused
PIF4IDR with an N-terminal mGFPmut2 and tested its phase separation ability in vitro. In the presence of
the macromolecular crowder PEG-8000 (10%), the recombinant mGFPmut2-PIF4IDR protein phase
separated into near-spherical condensates at a minimum concentration of 1 µM, and the granule size
increased as a function of the protein concentration (Fig. 1i). We further performed FRAP on these in
vitro-formed mGFPmut2-PIF4IDR condensates and discovered that they displayed poor liquidity at both
20 °C and 27 °C (Fig. 1j–l). These data suggest that PIF4 can form low-mobility condensates both in
vitro and in vivo, and its phase separation ability may not be affected within the ambient temperature
range.

PIF4 possesses an N-terminal TAD
Intrinsically disordered TADs of transcriptional activators have been reported to play a crucial role in
transcriptional condensate formation and subsequent gene expression46,63. However, a recent study
contended that TAD-mediated multivalent interactions, rather than the phase separation capacity of a
transcription factor, are essential for transcriptional activation64. We set out to investigate the
relationship between the PIF4 TAD, its phase separation propensity, and its function in
thermoresponsive gene expression.

The TAD of PIF3, the prototypical PIF protein, has been extensively characterized57,65. In the same
studies, some critical residues in the PIF4 TAD, such as Glu29, Trp32, Trp74, Phe84, Phe89, Phe93,
and Phe94, were identified by comparing it with the PIF3 TAD based on sequence homology. We
decided to comprehensively investigate the PIF4 TAD to extract precise sequence features for its
transactivation activity. We first mapped the PIF4 TAD using a yeast transactivation system. Three
fragments, PIF4A (1–78 a.a.), PIF4B (79–242 a.a.), and PIF4C (243–430 a.a.), were each fused to the
GAL4 DBD. Only the N-terminal 78 a.a. displayed strong transactivation activity in yeast
(Supplementary Fig. 1a). Further dissection of the PIF4A fragment revealed no transactivation activity
(Supplementary Fig. 1b).

To identify key residues responsible for PIF4’s transactivation activity in the N-terminal region, we
conducted alanine scanning mutagenesis across the first 91 a.a. of the PIF4 protein. Beginning with the
second residue, we generated 18 mutants (m1–m18) by substituting five consecutive residues with
alanines and tested their impact on transactivation activity in yeast. Using three reporters (AUR1-C,
HIS3, and LacZ), we identified four regions—m2 (7-11 a.a.), m5-m7 (22-36 a.a.), m13-m15 (62-76
a.a.), and m17-m18 (82-91 a.a.)—that showed a marked reduction in PIF4’s transactivation activity,
which we named TAD1 to TAD4, respectively (Supplementary Fig. 1c and Fig. 2a). TAD2 overlaps
with the APB domain, and several mutations (e.g., E29V and W32A) are known to disrupt phyB
interactions65. In contrast, mutations in TAD3 and TAD4, such as W74A, F84A, F89A, and FF93-
94AA, had little or no effect on phyB binding65.

We were particularly intrigued by m14 (67–71 a.a.) and m15 (72–76 a.a.), which overlap with the p53-
like ΦxxΦΦ motif identified in the PIF3 TAD (Supplementary Fig. 1c and Fig. 2b)57. The m14 region
contains three acidic residues, two of which (D68 and E70) are highly conserved among land plants, and
the m15 region harbors three conserved hydrophobic residues (V72, W74, and I75) (Fig. 2c). However,
substituting each hydrophobic residue in m15 did not affect PIF4’s transactivation activity in yeast
(Supplementary Fig. 1d). Therefore, we generated a mutated TAD variant (mTAD) by substituting the
three acidic residues in m14 and the three hydrophobic residues in m15 with alanines (Fig. 2c). Similar
to the m14 and m15 mutants, the mTAD variant drastically reduced PIF4’s transactivation activity in
yeast (Fig. 2b). We also assessed its effect on PIF4 function in planta by co-expressing the PIF4 protein
with a firefly luciferase reporter driven by the IAA19 promoter—a well-known PIF4 target. While wild-
type PIF4 significantly boosted luciferase expression, PIF4mTAD failed to do so when expressed at the
same level as the wild-type protein (Fig. 2d–g). Consequently, we chose to use the mTAD variant to
further explore how the PIF4 TAD contributes to phase separation and gene activation in plants.

PIF4 TAD mutations modestly affect PIF4 phase separation and its transcriptional activity in
thermomorphogenesis
We first compared the biophysical properties of condensates formed by wild‑type PIF4 and its mTAD
variant. When the proteins were expressed as mGFPmut2 fusions and incubated with 10% PEG‑8000,
PIF4mTAD still underwent phase separation (Fig. 3a). Nevertheless, mGFPmut2–PIF4mTAD produced
denser condensates with a smaller mean area, owing to a higher proportion of small granules (≤ 0.3 µm²)
and a scarcity of larger ones (≥ 1.0 µm²) compared with the wild‑type protein (Fig. 3b–d). A similar
pattern emerged in vivo: in tobacco nuclei, the phase‑separation propensity of PIF4mTAD, expressed as
an mGFPmut2 fusion, was diminished (Fig. 3e). Both the total number and the average size of nuclear
speckles decreased, with PIF4mTAD generating more small and fewer large speckles (Fig. 3f–h). The
mutation also heightened the diffuse nucleoplasmic signal (Fig. 3i). Collectively, substitutions in the
m14/m15 region partially compromise the phase‑separation capacity of PIF4 both in vitro and in vivo.

We further investigated whether the attenuated transactivation activity (Fig. 2) and reduced
phase‑separation propensity (Fig. 3a–i) of the mTAD variant would translate into impaired
thermomorphogenetic growth in Arabidopsis. Accordingly, we generated transgenic lines expressing
either wild‑type or mTAD PIF4, each with a C‑terminal mGFPmut2‑6His fusion driven by the native
PIF4 promoter in a pif4 mutant (SAIL_1288_E07) background. We obtained six PIF4pro::PIF4/pif4
and eight PIF4pro::PIF4mTAD/pif4 single‑insertion lines, which displayed a range of hypocotyl lengths
at 20 °C and 27 °C (Supplementary Fig. 2a). Because hypocotyl length correlates with PIF4 expression,
we plotted each line’s absolute hypocotyl length at 27 °C against its relative PIF4 transcript level
(Col‑0 = 1) and performed nonlinear regression to compare wild‑type and mTAD datasets
(Supplementary Fig. 2b). At equivalent expression levels, PIF4pro::PIF4mTAD/pif4 seedlings exhibited
slightly shorter hypocotyls than their wild‑type counterparts.

To refine this analysis, we selected two pairs of transgenic lines with comparable PIF4 transcript levels
(Supplementary Fig. 2b and Fig. 3j). In both pairs, PIF4 protein abundance at 27 °C was similar between
the mTAD and wild‑type lines (Fig. 3m). Nevertheless, thermomorphogenetic hypocotyl elongation was
partially compromised in the mTAD lines under both red and white light (Fig. 3j–l and Supplementary
Fig. 2c,d). Consistent with the modest hypocotyl shortening, thermo-induced expression of the PIF4
targets IAA19 and IAA29 trended lower in the mTAD lines, but only the reduction in IAA19 level within
the higher-PIF4 line pair (PIF4/pif4 #17 and PIF4mTAD/pif4 #13) reached statistical significance,
whereas changes in the lower-PIF4 pair (PIF4/pif4 #15 and PIF4mTAD/pif4 #10) and those in IAA29
expression across both pairs were not significant (Fig. 3n). Although these data demonstrate that the
mTAD substitution diminishes PIF4 function during thermomorphogenesis, the reduction in planta is
relatively modest, especially given that TAD activity was nearly abolished in yeast (Fig. 2b).

Acidic and hydrophobic residues in PIF4 TAD are crucial for its phase separation capacity
The observation that mutating the TAD of PIF4—a pivotal thermosensory transcriptional activator—
only had a modest impact on thermomorphogenesis is unexpected. Given the long‑recognized
length‑dependent modularity of TADs66,67, we asked whether the six alanine substitutions in PIF4mTAD
were simply insufficient to abolish its transactivation capacity in planta. Notably, the acidic and
hydrophobic character of the PIF4 TAD mirrors that of classical acidic TADs from plant, yeast, and
human activators42,68,69. According to the recently proposed “Acidic Exposure” model, acidic residues
alternate with hydrophobic residues within an acidic TAD, and electrostatic repulsion among the acidic
side chains exposes the hydrophobic residues to solvent, allowing co‑activator engagement68,69. We
therefore re‑evaluated the PIF4 TAD with the improved mechanistic predictor of acidic TADs that
tallies four charged residues (D, E, R, K) and three hydrophobic residues (W, F, L)70.

We dissected PIF4 into 39‑amino‑acid sliding windows, stepped by a single residue (Fig. 4a). For each
of the 392 tiles, we summed the counts of W, F, and L and calculated the net charge (D or E = −1; R or
K = +1). Applying the predictor, we found that tiles 51–70, spanning residues 51–109, met both
criteria—W + F + L ≥ 6 and −13 ≤ charge ≤ −8. Within this acidic TAD, acidic residues (D and E) are
interleaved with bulky hydrophobics (W, F, Y, L) (Fig. 4b). Analyses of PIF4 homologues from other
land plants revealed the same architectural motif despite low overall sequence identity (Supplementary
Fig. 3 and Fig. 4b).

To test the functional contribution of these residues, we created two mutants: mDE, in which six Ds and
four Es were replaced by Ns and Qs, respectively; and mWFYL, in which nine bulky hydrophobic
residues were substituted by As (Fig. 4b). Both variants curtailed PIF4IDR phase separation in vitro,
yielding more small granules (≤ 0.2 µm2) and fewer very large granules (> 1 µm2); the reduction was
stronger in mDE (Fig. 4c,d). Intriguingly, the overall granule density and mean size of mWFYL
granules still resembled those of the wild‑type protein (Fig. 4e,f). In vivo, however, both mDE and
mWFYL almost completely eliminated PIF4 nuclear speckle formation in N. benthamiana, with
mWFYL producing a more severe phenotype (Fig. 4g-j). These data suggest that both acidic and
hydrophobic residues in PIF4 TAD are crucial for its phase separation propensity.

The acidic and hydrophobic residues of the PIF4 TAD have distinct roles in
thermomorphogenesis
The strong reduction of nuclear speckles in the mDE and mWFYL variants (Fig. 4) made these two
mutants ideal for testing how PIF4 phase-separation capacity relates to its role in thermomorphogenesis.
We therefore created single-insertion PIF4pro::PIF4mDE and PIF4pro::PIF4mWFYL lines in the pif4
mutant background and compared their thermo-responsive phenotype with PIF4pro::PIF4 controls
(Fig. 5).

Surprisingly, PIF4mDE-mGFPmut2 protein was barely detectable in PIF4pro::PIF4mDE lines #24 and #26
(Fig. 5d). However, both lines accumulated PIF4 transcripts at levels between those in PIF4pro::PIF4
lines #15 and #25 (Fig. 5e), indicating that acidic residues in the PIF4 TAD are required for its protein
stability. As expected, both PIF4pro::PIF4mDE lines failed to rescue the pif4 thermomorphogenetic
defects as their hypocotyls remained as short as those of the pif4 mutant at 27 °C (Fig. 5a–c).
Consistently, thermo-induced expression of IAA19 and IAA29 was still suppressed in these
PIF4pro::PIF4mDE lines (Fig. 5f). Because the PIF4mDE-mGFPmut2 protein is unstable, these data do
not allow us to conclude whether acidic residues are directly required for PIF4’s transactivation activity
in thermomorphogenesis.

We next examined the mWFYL mutant, which showed a stronger in-planta defect in phase separation.
Two single-insertion PIF4pro::PIF4mWFYL lines, #24 and #12, accumulated protein amounts comparable
to PIF4pro::PIF4 lines #15 and #19, respectively (Fig. 5j). Surprisingly, neither PIF4pro::PIF4mWFYL
line showed any defects in thermomorphogenesis—both had similar hypocotyl length as their wild-type
counterparts at 27 °C (Fig. 5g–i). Their overall thermo-induced elongation ratios, however, were lower
than those of the corresponding wild-type lines (Fig. 5i), largely owing to their longer hypocotyls at
20 °C (Fig. 5h). Once again, thermo-responsive transcription of PIF4 target genes showed a mixed
pattern (Fig. 5k). In the higher-expression pair, they were significantly lower in PIF4pro::PIF4mWFYL
line #12 than PIF4pro::PIF4 line #19; whereas in the lower-expression pair, IAA19 and IAA29 levels in
PIF4pro::PIF4mWFYL line #24 were similar to, or even higher than, those in PIF4pro::PIF4 line #15
(Fig. 5k). Thus, although hydrophobic residues are important for PIF4 phase-separation propensity, they
appear largely dispensable for the thermosensory activation of genes driving thermomorphogenetic
hypocotyl elongation.

The basic region in PIF4 IDR is essential for its phase separation capacity
Collectively, our TAD-mutant analyses suggest that PIF4’s phase-separation capacity, as governed by
the TAD, is largely uncoupled from its transcriptional output and thermomorphogenetic function. To
probe which portion of the IDR (30–288 a.a.) drives PIF4 condensate formation, we extended our focus
beyond the TAD itself (51–109 a.a.), which constitutes <25 % of the IDR and is followed by an NLS as
well as the basic motif and first helix of the HLH domain. The key question is whether the TAD initiates
condensate nucleation or merely modulates assembly orchestrated elsewhere in the IDR. We therefore
set out to identify the minimal segment that is both necessary and sufficient for PIF4 condensate
formation.

We first performed in vitro phase‑separation assays using a series of N‑ and C‑terminal truncations of
PIF4IDR (Supplementary Fig. 4a). Remarkably, the extreme C‑terminus of PIF4IDR (244–288 a.a.) was
indispensable for condensate formation, whereas the N‑terminal segment containing the TAD (N0–N4)
failed to generate droplets in vitro (Supplementary Fig. 4b,c). By contrast, every C‑terminal fragment
(C1–C4) lacking the TAD readily produced speckles comparable to those formed by the full‑length
PIF4IDR (Supplementary Fig. 4b,c). These observations indicate that the TAD is insufficient on its own
to drive condensate assembly but could potentially modulate PIF4 phase separation. Moreover, the C4
segment (217–288 a.a.) is both necessary and sufficient to promote phase separation.

The C4 segment harbors three defined structural elements: an NLS peptide, the basic DNA‑binding
motif, and the first helix of the HLH dimerization/tetramerization domain (Fig. 6a). Although the basic
motif and its adjoining helix are highly conserved across land plants, the upstream sequence is markedly
diverse (Fig. 6a). Amino‑acid composition analysis of the C4 segment confirmed an unusually high
proportion of basic residues—arginine plus lysine together account for 20–25%, whereas aromatic
residues (W, Y, F) are virtually absent (Fig. 6a; Supplementary Fig. 5). Arginine is known to promote
phase separation through cation–π interactions with aromatic residues; recent studies further indicate
that its guanidinium group can engage in π–π stacking interactions that promote phase separation71,72.
We therefore hypothesized that the elevated arginine content in C4 is critical for PIF4’s propensity to
phase‑separate.

To test this idea, we generated four alanine‑substitution variants of C4: mALL (all 17 basic residues),
mNLS (5 basic residues within the NLS), mBH (12 basic residues spanning the basic and helix regions),
and mB (9 residues confined to the basic motif) (Fig. 6a). These KR substitutions impaired
mGFPmut2‑PIF4C4 phase separation in a dose‑dependent manner—mNLS, mBH, and mB modestly
reduced droplet size, whereas mALL completely abolished condensate formation (Fig. 6b–f). Next, we
evaluated the same mutations in full‑length PIF4 expressed in tobacco. As expected, mALL and mNLS
markedly diminished mGFPmut2‑PIF4 nuclear localization and were therefore excluded from further
comparison (Fig. 6g). Both mBH and mB significantly lowered the number of nuclear speckles but
tended to coalesce into larger condensates (Fig. 6g–j). Collectively, these results confirm that basic
residues are critical for PIF4’s capacity to phase separate.

Basic residues in the basic motif and first helix of the PIF4 bHLH domain are indispensable for
thermomorphogenesis
Observing that basic residues in the C4 segment are required for PIF4 phase separation, we generated
single-insertion PIF4pro::PIF4mBH and PIF4pro::PIF4mB lines in the pif4 background to assess their
contribution to thermomorphogenesis (Fig. 7).

We selected two PIF4pro::PIF4mBH lines (#3 and #15) that accumulated substantially more PIF4 protein
than the PIF4pro::PIF4 control lines (#15 and #17) (Fig. 7d). Despite these high protein levels, both
PIF4pro::PIF4mBH lines displayed hypocotyl lengths similar to the pif4 mutant at 20 °C and 27 °C (Fig.
7a–c), indicating that the 12 basic residues spanning the basic motif and the first helix of the bHLH
domain are essential for PIF4-mediated hypocotyl elongation at warm temperatures. By contrast, two
PIF4pro::PIF4mB lines (#12 and #2), which expressed PIF4 at levels comparable with their wild-type
counterparts (#19 and #25), largely rescued the thermomorphogenetic defect: their hypocotyls were as
long as—if not longer than—those of the control lines (Fig. 7e–h).

These results yield three insights. First, PIF4’s phase-separation capacity is not strictly required for
thermomorphogenesis, because PIF4mB–mGFPmut2 remains functional despite its markedly reduced
condensate formation (Fig. 6). Second, PIF4’s DNA binding ability per se might be dispensable, as the
basic motif, known for its canonical DNA-binding function in bHLH transcription factors, can be
mutated without loss of function in thermomorphogenesis. Third, the failure of PIF4mBH–mGFPmut2,
but not PIF4mB–mGFPmut2, to rescue the pif4 defects in thermosensory growth points to the three basic
residues in the first helix as crucial for PIF4’s thermomorphogenetic activity. This first conclusion is
reinforced by our PIF4 TAD-mutant analyses, and we therefore pursued the latter two points in greater
detail.

Disrupting PIF4 DNA binding modestly affects thermomorphogenesis
The basic motif of bHLH transcription factors mediates DNA binding50,73,74, and several arginine
residues in PIF4’s basic region—R254, R257, R269, and R270—are critical for PIF4’s affinity to target
promoters51. It is therefore striking that replacing nine basic residues in PIF4’s basic motif (variant mB)
scarcely affected its thermomorphogenetic activity (Fig. 7e–h).

To probe this further, we drew on work with PIF3, the prototypic PIF protein, where substituting two
conserved arginines in the basic region (R353Q/R355Q) abolished DNA binding75. These two arginines
are highly conserved in the PIF family and align with R267 and R269 in PIF4 (Supplementary Fig. 5c).
Because multivalent IDR interactions often scale with length, we reasoned that converting R267 and
R269 to glutamines (hereafter RRQQ) would eliminate DNA binding while minimally perturbing phase
separation. Consistent with this idea, the RRQQ substitutions, like mB, completely abolished binding of
the PIF4 bHLH domain to a G-box (CACGTG) fragment from the IAA19 promoter in EMSA
(Supplementary Fig. 6i). In vitro, the RRQQ version of mGFPmut2-PIF4C4 protein formed condensates
indistinguishable in density and size from the wild-type protein (Supplementary Fig. 6a–d). When
expressed as the full-length PIF4 in tobacco, however, mGFPmut2-PIF4RRQQ produced fewer and larger
nuclear speckles, but the effect was milder than mGFPmut2-PIF4mB (Supplementary Fig. 6e–h; Fig. 6g–
j).

We analyzed two single-insertion PIF4pro::PIF4RRQQ lines (#22 and #3), whose PIF4 abundance laid
between that of PIF4pro::PIF4 lines #15 and #17 (Supplementary Fig. 6m). Both PIF4pro::PIF4RRQQ
lines largely rescued the pif4 thermomorphogenetic defect, exhibiting hypocotyl lengths intermediate
between the two controls at 20 °C and 27 °C (Supplementary Fig. 6j-l). Thermo-induced expression of
the PIF4 targets IAA19 and IAA29 trended lower in PIF4pro::PIF4RRQQ lines, but the differences were
not significant; only IAA29 expression in control line #17 exceeded that in both PIF4pro::PIF4RRQQ
lines (Supplementary Fig. 6n). Together, these data indicate that PIF4’s DNA-binding activity makes
only a minor contribution to thermomorphogenesis. These counterintuitive results suggest that PIF4’s
DNA-association and subsequent transcriptional regulation might be assisted by the DNA-binding and
transactivation activities of its close partners (e.g., other PIFs).

The oligomerization ability is central to PIF4-mediated thermomorphogenesis
Our TAD‐ and basic-region analyses indicate that PIF4 phase separation capacity is largely dispensable
for thermosensory hypocotyl growth. By contrast, the divergent behavior of the mBH and mB variants
points to three basic residues in the first helix of the bHLH domain—R272, R276, and K278—as critical
determinants of PIF4 thermomorphogenetic function. R276 and K278 lie within one of the two dimer–
dimer interfaces that assemble the PIF4 bHLH tetramer51. Previous work showed that extensive
substitution of these interfaces forces the domain to exist only as dimers. We therefore hypothesized that
the inability of the mBH variant to rescue the pif4 mutant stems from a failure to oligomerize.

To test this idea, we purified wild-type and mutant variants of MBP–PIF4bHLH and performed a
Bis(sulfosuccinimidyl) suberate (BS3) crosslinking assay. Like the wild-type MBP–PIF4bHLH, the mB
version, which alters only the basic motif, showed both dimers and high-molecular-weight multimers,
whereas the multimer band was barely visible in the mBH version, which additionally targets the first
helix (Fig. 8a). These results confirm that the basic residues in the first helix underpin PIF4
oligomerization and that the loss of oligomerization underlies the functional defect of the mBH variant.

The HLH domain of PIF4 can also be used for its hetero-oligomerization with other PIFs50–52. Through
GST pulldown assays, we further confirmed that the mBH version, but not the mB variant, dramatically
reduced the interaction between PIF4 and other PIF proteins (Supplementary Fig. 7a). We therefore
hypothesized that the partnership between PIF4 and other PIFs was the reason that PIF4 variants with
weakened PIF4 TAD activity (e.g., mTAD and mWFYL) or DNA-binding (mB and RRQQ) could still
largely complement the pif4 null mutant (Fig. 3j–n, Fig. 5g–k, Fig. 7e–h, and Supplementary Fig. 6j–n).
If this is true, removing close partners would unmask the importance of these functions of PIF4 in
thermosensory gene expression. We decided to use the mTAD version of PIF4 as an example to test this
model. We first confirmed that mTAD mutations didn’t affect PIF4 binding affinity to other PIFs
(Supplementary Fig. 7b), suggesting that the mTAD variant could still hetero-oligomerize with other
PIFs. Next, we generated single‑insertion PIF4pro::PIF4 and PIF4pro::PIF4mTAD lines in the pifq
(pif1 pif3 pif4 pif5 quadruple) mutant background (Supplementary Fig. 8a). Plotting absolute hypocotyl
length at 27 °C against relative PIF4 transcript abundance (Col‑0 = 1) revealed a clear dosage response
in PIF4pro::PIF4/pifq seedlings, whereas PIF4pro::PIF4mTAD/pifq seedlings remained as short as pifq
irrespective of the PIF4mTAD transgene level (Supplementary Fig. 8b). We compared two lines each from
PIF4pro::PIF4mTAD/pifq (#2 and #23) and PIF4pro::PIF4/pifq (#24 and #4). Despite accumulating
higher levels of PIF4 protein, the PIF4pro::PIF4mTAD/pifq seedlings were drastically shorter than their
wild‑type counterparts at 27 °C (Fig. 8b–d). Consistently, warm-temperature induction of the PIF4
targets IAA19 and IAA29 was abolished in both PIF4pro::PIF4mTAD/pifq lines (Fig. 8e). Collectively,
these data support a partner-dependent mechanism in which oligomerization competence is critical for
robust thermomorphogenetic output, while PIF4’s transactivation and DNA-binding contributions can
be supplied in trans when partners are available (Fig. 8f).

Methods
Plant materials and growth conditions
The Arabidopsis mutants pif4-2 (SAIL_1288_E07) and pifq have been previously described93.
Homozygosity was confirmed by PCR before the seeds were used for phenotyping and Agrobacterium-
mediated floral dipping. Seeds were surface sterilized with bleach (3% sodium hypochlorite) for 8 min
before being plated on half-strength sucrose-free Murashige and Skoog (1/2 MS0) media supplemented
with Gamborg’s vitamins (Caisson Laboratories, North Logan, UT), 0.5 mM MES (pH 5.7), and 0.8%
(w/v) agar (Caisson Laboratories, North Logan, UT). Seeds were stratified in the dark at 4 °C for 3-5
days to synchronize germination before treatment under specific light and temperature conditions in
LED chambers (Percival Scientific, Perry, IA). Fluence rates of light were measured using an Apogee
PS200 spectroradiometer (Apogee Instruments Inc., Logan, UT).

Hypocotyl measurements
Seeds were plated on the surface of ½ MS0 media and grown at either 20 °C or 27 °C under continuous
red light (50 µmol m-2 s-1) for 96 hours. More than fifteen seedlings from each line were placed on
transparent film and scanned using an Epson Perfection V700 photo scanner. Hypocotyl length was
measured using NIH ImageJ software (http://rsb.info.nih.gov/nih-image/). The percent increase (PI) in
hypocotyl length was calculated as the percentage change observed at 27 °C relative to the
corresponding measurement at 20 °C. The relative response of each mutant was defined as the
percentage of its PI value normalized to that of the Col-0 wild type. At least three replicates were used
to calculate the mean and standard deviation of each relative response. Violin and box plots were
generated using Prism 10 (GraphPad Software, San Diego, CA).

Plasmid constructions
All the primers used for cloning are listed in the Supplementary Table 1. PCR amplifications were
performed using the Q5 High-Fidelity DNA Polymerase and the ligation reactions with the NEBuilder
HiFi DNA Assembly Master Mix (New England Biolabs Inc., Ipswich, MA). To generate various
mGFPmut2-PIF4 recombinant proteins, the coding sequence (CDS) of mGFPmut2, the GS-linker
(GAPGSAGSAAGGSG), and the wild-type or mutant PIF4 were cloned into pET28a vectors between
EcoRI and XhoI. To generate MBP-PIF4bHLH recombinant proteins, the wild-type or mutant coding
sequence of the PIF4 bHLH domain was amplified and cloned into the pMAL-C2X vector between
EcoRI and BamHI. For transient protein expression and luciferase assay, the sequence of mGFPmut2-
GS-PIF4 was cloned into pCHF1 or pCHF3 between BamHI and SalI. For the luciferase assay, the
luciferase gene and an RbcS terminator sequence were cloned into pJHA212B between PstI and HindIII,
and a 2-kb IAA19 promoter was added between EcoRI and SalI. Mutations or fragments of PIF4 CDS
were cloned into pBridge between EcoRI and SalI for alanine scanning and transactivation assay. To
generate transgenic lines, a 2-kb promoter region of PIF4 was fused with PIF4 CDS (wild-type and
mutant versions) and cloned into pJHA212H between EcoRI and BamHI along with a C-terminal
(PT)4 linker, mGFPmut2, and a His-tag. To generate in vitro transcribed/translated PIF4 proteins, the
wild-type or mutant coding sequence of PIF4 was amplified and cloned into the pCMX-PL2-NHA
vector between EcoRI and XmaI. To generate GST-PIF3 and GST-PIF5 recombinant proteins, the
coding sequence of PIF3 and PIF5 were cloned into pET42b vectors between BamHI and HindIII.

Recombinant protein expression and purification
For recombinant protein expression, the aforementioned pET28a or pMAL-C2X plasmids were
transformed into BL21 (DE3) Rosetta strains of E. coli by electroporation. The expression of
recombinant proteins cloned into pET28a was induced at 37 °C for 3 h with 0.5 mM IPTG. Cells were
harvested and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 mM imidazole, and 2
mM PMSF) using a Pulse 150 Ultrasonic Homogenizer with Φ6 probe (Benchmark Scientific,
Sayreville, NJ). The supernatant was harvested and incubated with lysis buffer-equilibrated Ni-NTA
His-Pur agarose (ThermoFisher Scientific, Waltham, MA) for 1 h at 4 °C. The protein-agarose slurry
was packed in a gravity-flow column (Bio-Rad Laboratories, Hercules, CA) and washed with 3 volumes
of wash buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 25 mM imidazole) and eluted using
elution buffers with increasing imidazole concentrations (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 50-
250 mM imidazole, and 1 mM DTT). Eluates were combined based on SDS-PAGE results, immediately
concentrated and desalted in a storage buffer (50 mM Tris-HCl, 125 mM NaCl, 10% glycerol, and 1
mM DTT) using 30K Amicon Ultra centrifugal filters (Sigma Aldrich Inc., St. Louis, MO), flash-frozen
in liquid nitrogen, and stored at -80 °C until use. Protein concentrations were measured using the
Bradford assay and verified by SDS-PAGE.

For proteins cloned into pMAL-C2X, the initial purification followed the same Ni-NTA His-tag
protocol as described above. After the eluates were desalted, MBP-tagged proteins were further purified
using amylose resin according to the manufacturer’s instructions (New England Biolabs Inc., Ipswich,
MA). Briefly, the protein-resin was packed into a gravity-flow column, washed with wash buffer (20
mM Tris-HCl, pH 7.5, 150 mM NaCl), and the protein was eluted with the same wash buffer containing
10 mM maltose. Final eluates were pooled, and protein quality was verified by SDS-PAGE after being
quantified using the Bradford assay.

In vitro droplet formation
Purified protein samples were diluted to the required concentrations and mixed with the respective
volume of droplet formation buffer (50 mM Tris-HCl, pH 7.5, 125 mM NaCl, 10% glycerol, 10% PEG-
8000, and 1 mM DTT). Imaging was performed in a glass-bottom 384-well plate (Cellvis, Mountain
View, CA) or on a glass slide (Fisherbrand, Pittsburgh, PA) using a Leica SP8 confocal microscope
(Leica Microsystems Inc., Buffalo Grove, IL). All the images were taken at the bottom-most plane (on
the surface of the 384-well plate or slide). Excitation and emission wavelengths for mGFPmut2 were
488 nm and 500-550 nm, respectively.

In planta condensate formation
Binary constructs based on the pCHF1 vector carrying PIF4 coding sequences or its mutant variants
were transformed into the GV3101 Agrobacterium strain by electroporation and screened on LB media
containing 20 µg/mL gentamycin, 25 µg/mL rifampicin, and 100 µg/mL spectinomycin. Individual
colonies were inoculated to grow a small-scale culture, which was harvested at an OD of 0.6,
600
resuspended in infiltration buffer (10 mM MES, pH 5.7, 10 mM MgCl ), and treated with 150 µM
2
acetosyringone for 2-4 h. The suspension was injected into tobacco leaves and incubated at 20 °C for
36-48 h. For temperature treatments, tobacco plants were first incubated at 20 °C for 24 h, and then
transferred to 28 °C for another 12 h. A portion of the leaf near the injection site was cut, mounted in
water, and imaged using the 40× immersion lens on the Leica SP8 confocal microscope. Excitation and
emission wavelengths for mGFPmut2 were 488 nm and 500-550 nm, respectively. A minimum of 4
leaves with at least 5 nuclei per leaf were examined for PIF4 condensate formation.

Fluorescent Recovery After Photobleaching (FRAP) assays
FRAP experiments were performed using the Leica SP8 confocal microscope with a 40× water-
immersion objective. The GFP signal was bleached using a 488 nm laser (100% laser power). For in
vitro samples, bleaching was performed with 2-4 iterations, while for tobacco samples, 8-10 iterations
were used to ensure sufficient bleaching. Fluorescence recovery was recorded every 10 seconds for 20
time points. Recovery curves were generated using the ImageJ software.

Luciferase assay in Nicotiana benthamiana
For the luciferase assay, Agrobacterium strains carrying pCHF1/PIF4-(PT)4-mGFPmut2-6His and
pJHA212B/IAA19pro::LUC-RbcSter constructs were mixed and co-infiltrated into tobacco leaves,
using the empty pCHF1 and pJHA212B/IAA19pro::LUC-RbcSter as a control. After 2 days of
incubation, the infiltrated leaves were cut and sprayed with 3 mM luciferin substrate. Luminescence
signals were detected using an Azure C600 Advanced Imaging System (Azure Biosystems, Dublin, CA,
USA), and the signal strength was quantified using the ImageJ software.

Yeast Transactivation Assay
Yeast transactivation and alanine scanning assays were conducted using either Y2HGold yeast strains
(TaKaRa Bio USA, San Jose, CA) harboring a pBridge bait vector or diploid yeast strains obtained by
mating Y2HGold (bait) with Y187 (prey) strains, the latter carrying the pGADT7 vector (TaKaRa Bio
USA, San Jose, CA). Overnight cultures were adjusted to an OD of 0.2 and subjected to tenfold serial
600
dilutions. A 10-µL aliquot from each dilution was spotted onto selective synthetic dropout media—
Y2HGold onto SD/-Trp media, and diploid strains onto SD/-Trp/-Leu (± 250 ng/mL Aureobasidin A)
and SD/-Trp/-Leu/-His (+ 10 mM 3-Amino-1,2,4-triazole) media. Plates were incubated at 30 °C, and
colony growth was documented on the third day.

For liquid β-galactosidase assays, diploid cells were generated by mating Y2HGold and Y187 strains
and selected on SD/-Trp/-Leu plates. These cells were then grown overnight in liquid SD/-Trp/-Leu
medium, and β-galactosidase activity was quantified using ortho-nitrophenyl-β-galactoside (ONPG) as
the substrate, following the Yeast Protocols Handbook (TaKaRa Bio USA, San Jose, CA).

Generation of transgenic lines
To generate wild-type and mutant versions of PIF4pro::PIF4-mGFPmut2 transgenic lines, the pif4-2
and pifq mutant plants were transformed with wild-type or mutant versions of
pJHA212H/PIF4pro::PIF4-(PT)4-mGFPmut2-6His plasmids using the Agrobacterium-mediated floral
dip method. Positive transformants were selected on the ½ MS0 medium containing 50 µg/mL
hygromycin. At least six independent lines that segregated approximately 3:1 for hygromycin-resistance
in the T2 generation were identified, and at least two lines were selected for this study based on the
transgene expression level. For all experiments, T3 self-progenies of homozygous T2 plants were used.

RNA extraction and quantitative reverse transcription PCR
50–100 mg of seedlings were used to extract total RNA using the Quick-RNA Miniprep Kit (Zymo
Research, Irvine, CA), with on-column DNase I treatment. First-strand cDNA synthesis was carried out
using 0.5-2.5 µg of total RNA using the SuperScript III Reverse Transcriptase and the Oligo(dT)20
primer (Thermo Fisher Scientific, Waltham, MA). For qPCR, diluted cDNA was mixed with 2×
Universal SYBR Green Fast qPCR Mix (ABclonal Technology, Woburn, MA) along with gene-specific
primers listed in Supplementary Table 2. Triplicates of each RT-qPCR reaction were performed using
the Quant Studio 3 (Thermo Fisher Scientific, Waltham, MA). Gene expression was normalized to
PP2A transcript levels and quantified using the 2-ΔΔCt method. Final data were visualized as bar graphs
using Prism 10 software (GraphPad Software, San Diego, CA).

Protein extraction and immunoblotting
For immunoblot analyses, total protein was extracted from 4-day-old seedlings as previously
described55. In brief, 200 mg of fresh seedlings were flash frozen and ground to a fine powder using
sterile wooden pestles and vortexed in three volumes of extraction buffer containing 100 mM Tris-HCl
(pH 7.5), 100 mM NaCl, 5 mM EDTA, 5% SDS, 20% glycerol, 20 mM DTT, 40 mM β-
mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride, 40 µM MG115 (Apexbio Technology LLC,
Houston, TX), 40 µM MG132 (Cayman Chemical, Ann Arbor, MI), 1× phosphatase inhibitor cocktail 2
(Thermo Fisher Scientific), 1× phosphatase inhibitor cocktail 3 (MilliporeSigma), 1× EDTA-free
protease inhibitor cocktail (MilliporeSigma), and 0.01% bromophenol blue. Protein extracts were then
boiled for 10 minutes and clarified by centrifugation at 20,000 × g for 10 minutes.

Cleared protein samples were separated via SDS-PAGE, transferred to nitrocellulose membranes at 70V
for 1-2 h, probed with the indicated primary antibodies overnight, and then incubated with a 1:5000
dilution of horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (Bio-Rad
Laboratories, 1706515). Primary antibodies, including polyclonal rabbit anti-PIF4 antibodies (Agrisera,
AS163157 and Abiocode, R2534-1) and polyclonal rabbit anti-RPN6 antibodies (Enzo Life Sciences,
BMLPW8370-0100), were used at 1:1000 dilutions. Signals were detected via chemiluminescence using
a SuperSignal kit (Thermo Fisher Scientific, Waltham, MA) and an Azure C600 Advanced Imaging
System (Azure Biosystems, Dublin, CA).

Electrophoretic mobility shift assay (EMSA)
5′-biotin-labeled complementary oligonucleotides corresponding to the G-box-containing region of the
IAA19 promoter were synthesized (Sigma-Aldrich, St. Louis, MO, USA). The wild-type probe sequence
was 5′–tatttcatataatttCACGTGgcccaacttgtttct–3′, and the mutated probe carried a disrupted G-box
motif: 5′–tatttcatataatttACAACAgcccaacttgtttct–3′. To generate double-stranded DNA probes,
oligonucleotides were annealed by heating at 95 °C for 5 min, followed by slow cooling to room
temperature. EMSAs were performed using the LightShift Chemiluminescent EMSA kit (Thermo Fisher
Scientific, Waltham, MA). Briefly, binding reactions (20 μL total volume) were carried out in binding
buffer (2 μL 10× binding buffer, 5 mM MgCl , 1 mM EDTA, 0.05% NP-40, 5% glycerol, 50 ng
2
poly(dI-dC)), 1–2 μg purified protein, and 20 fmol of biotin-labeled probe. The reaction mixture was
incubated at 4 °C for 20–30 min. Binding reactions were loaded on a 6% native polyacrylamide gel and
subsequently transferred to a nylon membrane (Merck Millipore, Burlington, MA). Signal was detected
using an Azure C600 Advanced Imaging System (Azure Biosystems, Dublin, CA).

BS3 crosslinking assay
Purified protein samples were buffer exchanged into a crosslinking-compatible buffer (20 mM HEPES,
pH 7.5, 150 mM NaCl) using 30K Amicon Ultra centrifugal filters (Sigma Aldrich Inc., St. Louis, MO).
Bis(sulfosuccinimidyl) suberate sodium salt (BS3) (Thermo Fisher Scientific, Waltham, MA) was
freshly prepared as a 25 mM stock solution in PBS immediately before use. For crosslinking, the protein
was mixed with 0.25 mM BS3 and incubated at 4 °C for 2 h. The reaction was quenched by adding 1 M
Tris-HCl (pH 7.5) and further incubated at room temperature for 15 minutes. Samples were mixed with
4× non-reducing SDS loading buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 40% Glycerol), subjected
to SDS-PAGE, and visualized with Coomassie blue staining.

GST pulldown assay
The prey proteins (PIF4, PIF4mTAD, PIF4mB, and PIF4mBH) were synthesized in vitro using the PURExpress In
Vitro Protein Synthesis Kit (New England Biolabs Inc., Ipswich, MA) according to the manufacturer’s
instructions. Recombinant bait proteins (GST, GST-PIF3, and GST-PIF5) were expressed in E. coli strain BL21
and purified using Glutathione Sepharose 4B beads (Cytiva, Marlborough, MA). Purification was carried out by
incubating bacterial lysates with the beads for 1 h at 4 °C. After incubation, the beads were
washed four times with wash buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) and then equally transferred to
new tubes. Prey proteins were mixed with the bait-beads complex and incubated in 500 µL binding buffer (20
mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1% DMSO) for another 2 h at 4 °C.
Subsequently, the beads were washed four times with the wash buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl,
1 mM EDTA, 1 mM EGTA, 1% DMSO, 2 mM DTT and 0.1% Nonidet P-40) to minimize nonspecific binding.
The pulled-down proteins were eluted with SDS loading buffer at 100 °C for 10 min and separated by SDS-
PAGE. Input and eluted prey proteins were subjected to immunoblots using anti-HA antibody (Abcam,
Cambridge, MA, USA). Recombinant bait proteins were detected by Coomassie Brilliant Blue staining.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Xiong et al., reported that PIF4 oligomerization but not its DNA-binding or transactivation activity is necessary for its role in thermomorphogenesis. I appreciate that the authors have generated so many mutated forms of PIF4 for detailed investigations. The conclusion of this report is novel and inspiring, contrary to conventional thinking, and holds significant implications for future research. The experiment is well designed and the writing is perfect. I just have a few comments.\n1. As we know, different PIF members have both overlapped and non-overlapped functions. For example, PIF1 but not PIF4 controls seed germination. In the last section of this manuscript, the authors simply transformed PIF4 and PIF4 mutated form in pifq for phenotyping. Is there any evidence to show that the mutated form of PIF4 could not interact with all the PIF members? Is it possible to tell the DNA binding specificity of each PIF members if PIF4 acts as a molecular glue?\n2. I am wondering whether the argued PIF4 molecular glue is a general mechanism not only in thermomorphogesis but also in other PIF4 regulated physiological events, such as photomorphogenesis, flowering, stomata development etc. It is easy to compare other phenotypes with the mentioned transgenic complementation lines.\n3. As argued, phase separation seems not required for PIF4 function in thermomorphogesis. But what is the biological significance of PIF4 condensation? Whether PIF4 interacts with other PIF members to co-condensate in the same droplet?", "reviewer_2": "The authors examined which domains of PIF4 are required for condensate formation and showed that the ability to form condensates is not essential for PIF4’s thermomorphogenetic function. They also tested whether PIF4’s transactivation and DNA-binding activities are required and concluded that they are not. Instead, the authors propose that heterodimerization/oligomerization is necessary. I have a few comments.\n1. Fig. 1b–1d. There are only six circles for 20 °C and four circles for 27 °C. The authors did not describe what these circles represent, but I presume they indicate individual nuclei. Six or four nuclei are too few, especially considering that these are from agro-infiltrated samples. Moreover, agro-infiltrated leaves—and even cells within the same leaf—exhibit substantial variation. The authors need to drastically increase both the number of nuclei analyzed per infiltrated leaf and the number of independent infiltrated leaves. Statistical differences can only be claimed after examining sufficiently large sample sizes. The same concern applies to other figures.\n2. The authors concluded from the FRAP assay (Fig. 1e–1f) that PIF4 forms gel-like condensates in vivo. However, the cytosolic GFP fluorescence signal is also not recovered (Fig. 1e), which calls their conclusion into question.\n3. It is difficult to follow how the authors identified TAD1 and TAD2. According to Fig. 1c, m2 (a.a. 7–11) fails to activate transcription, while m16 (a.a. 77–81) retains transcriptional activity. A straightforward interpretation of Fig. S1c suggests four separate TADs (m2, m5–7, m14–15, and m17–18), yet the authors concluded that there are two. Please clarify how the authors arrived at two TADs from these data.\n4. Quail’s group has already identified PIF4 TADs (Dalton et al., 2016), which are virtually identical to those reported here. Thus, it is not accurate to state that “critical features of the PIF4 TAD remain unknown (line 167).” Oddly, the authors cite Dalton et al. (2016) only regarding the lack of interaction of point mutants with phyB. Please revise the relevant paragraphs on PIF4 TAD identification to first acknowledge and cite this prior work.\n5. Although the mTAD version identified in Fig. 2 lost TAD activity in transient assays, it still activated target gene expression in stable transgenic Arabidopsis (Fig. 3). Therefore, the data in Fig. 2 cannot be used to conclude that TAD activity is not required for function.\n6. The authors showed that mTAD (Fig. 3) and mDE/mWYFL (Fig. 4) form primarily smaller granules both in vitro and in vivo. However, deletion of these regions did not affect granule formation in vitro (Fig. S4). Where does this discrepancy arise? Please clarify.\n7. The authors concluded that “These observations indicate that the TAD functions primarily as a regulator of PIF4 phase separation (line 321).” Since deletion of TAD regions does not affect granule formation (Fig. S4), how was this conclusion drawn? Furthermore, what do the authors mean by “regulator”?\n8. mWFYL formed condensates in vitro (Fig. 4c) but not in vivo (Fig. 4g). Similarly, the RRQQ mutation affected condensate formation differently in vitro versus in vivo. These results suggest that the capacity to form condensates differs between in vitro and in vivo conditions. Thus, conclusions should be drawn more cautiously, and the basis for each conclusion should be explicitly stated. For example, the claim that N-terminal regions are non-essential for condensate formation is supported by in vitro but not in vivo data. Shouldn’t the authors temper their conclusions in light of Fig. 4c/4g?\n9. The authors concluded that “PIF4’s transactivation and DNA-binding activity make only a minor contribution.” However, this interpretation requires more caution. First, as the authors note, PIF4 can heterodimerize/oligomerize with other bHLH proteins. In vivo, PIF4 mutant alleles (mTAD, mB, RRQQ) could dimerize/oligomerize with other PIFs (PIF1/3/4/5/7), thereby binding DNA and activating gene expression. Indeed, the authors showed that mTAD is functional in the pif4 mutant background (Fig. 3) but not in the pif1/3/4/5 background (Fig. 8). A simple explanation is that transactivation activity is supplied by heterodimerized/oligomerized PIFs, which suggests that transactivation activity is in fact necessary. The same reasoning applies to DNA-binding activity. To test this, please also include PIF7, which is known to play an important role in thermomorphogenesis.\n10. The authors suggest that PIF4’s HLH domain might possess unique properties that support heterotypic oligomerization with non-PIF cofactors during thermomorphogenesis. However, as I remember, a previously report (Kim et al(?) (2024) Plant Cell) demonstrated that the C-terminal domains of PIF1 and PIF4 are interchangeable, indicating that unique HLH characteristics are unlikely."}, "author_response": "We want to thank the reviewers for their insightful comments and suggestions, which helped us improve the quality and readability of our manuscript. We have addressed all the questions and made changes in the text, figures, figure legends, and supplementary data in the revised manuscript.\nReviewer #1 (Remarks to the Author):\nXiong et al. reported that PIF4 oligomerization but not its DNA-binding or transactivation activity is necessary for its role in thermomorphogenesis. I appreciate that the authors have generated so many mutated forms of PIF4 for detailed investigations. The conclusion of this report is novel and inspiring, contrary to conventional thinking, and holds significant implications for future research. The experiment is well designed and the writing is perfect. I just have a few comments.\n1. As we know, different PIF members have both overlapped and non-overlapped functions. For example, PIF1 but not PIF4 controls seed germination. In the last section of this manuscript, the authors simply transformed PIF4 and PIF4 mutated form in pifq for phenotyping. Is there any evidence to show that the mutated form of PIF4 could not interact with all the PIF members? Is it possible to tell the DNA binding specificity of each PIF member if PIF4 acts as a molecular glue?\nResponse: Thank you for this thoughtful question. We fully agree that PIFs exhibit both overlapping and distinct functions. It has been well known that PIF4 plays a central role in thermomorphogenesis, as the pif4 single knockout leads to severe defects in thermosensory growth—thermo-induced hypocotyl and leaf petiole elongation is dramatically hampered. Accumulating evidence suggests that other PIFs, including PIF1, PIF3, PIF5, and PIF7, also contribute to Arabidopsis thermomorphogenesis1–3. Consistent with this, our data support a redundancy-at-the-complex model for thermomorphogenesis: PIF4 is indispensable in this pathway (the pif4 null shows strong thermosensory defects), yet its intrinsic TAD/DBD activities can be functionally supplemented by partner PIFs when oligomerization is intact. Accordingly, PIF4 variants with impaired TAD (mTAD and mWFYL) or DBD/basic region (mB and RRQQ) largely complement the pif4 single mutant’s hypocotyl phenotype (Fig. 3j–n; Fig. 5g–k; Fig. 7e–h; Supplementary Fig. 6j–n), while the mTAD allele fails in the pifq background (Fig. 8b–e), where key partners are absent. These findings indicate that PIF4’s oligomerization is absolutely required, whereas its own TAD/DBD can be dispensable when partner PIFs are present.\nThe reviewer asked a good question about whether mTAD affects PIF4 interaction with other PIFs. We realize that a prerequisite assumption of the above model is that the mBH variant disrupts not only PIF4 homo-oligomerization but also its hetero-oligomerization with other PIFs, whereas the mB and mTAD mutations do not affect PIF4’s oligomerization ability. Because mB and mTAD complemented pif4 (where partner PIFs are present), we inferred that HLH-mediated interactions were not affected. To test this directly, we performed GST pulldown assays and confirmed that neither mB nor mTAD affected PIF4’s interactions with PIF3 and PIF5, while the mBH variant dramatically decreased PIF4’s binding affinity to both PIFs. These results are summarized in Supplementary Fig. 7 and discussed in the Results section (lines 429–440).\nAbout the last question, by describing PIF4 as a “molecular glue,” we do not mean that its DNA binding or transactivation is irrelevant; rather, partner PIFs can supply sufficient TAD/DBD function within the oligomer, rendering PIF4’s own TAD/DBD conditionally dispensable in Arabidopsis. The “molecular glue” term emphasizes the essentiality of PIF4’s oligomerization activity. Disentangling the DNA-binding specificities of individual PIFs in this cooperative context will require genomic assays (e.g., ChIP-seq or CUT&RUN) in thermomorphogenetic conditions, ideally combined with perturbations that selectively disrupt oligomerization interfaces. We outline these needs and predictions in the Discussion section (lines 539–542).\n2. I am wondering whether the argued PIF4 molecular glue is a general mechanism not only in thermomorphogesis but also in other PIF4 regulated physiological events, such as photomorphogenesis, flowering, stomata development etc. It is easy to compare other phenotypes with the mentioned transgenic complementation lines.\nResponse: Thanks for raising this interesting question. Indeed, PIF4’s important role is not restricted to thermomorphogenesis, including thermosensory hypocotyl and petiole elongation, early flowering, and reduced stomata density at warm temperatures; it also plays a vital role in light signaling, such as shade avoidance, photomorphogenesis, and skotomorphogenesis, as well as senescence, immunity, salt and drought tolerance, and hormone crosstalk. In other words, PIF4 functions as a master regulator that integrates various environmental and hormonal signals in modulating plant growth and development. We are also curious to know whether our PIF4 molecular glue model could be applied to other pathways regulated by PIF4.\nIn fact, we did have the data supporting PIF4’s molecular glue function in photomorphogenesis in the original submission. Firstly, the pif4 null mutant displayed a significantly short hypocotyl phenotype in the red light (50 µmol m-2 s-1) at 20 °C (Fig. 3j, k), consistent with its essential function in phyB-mediated photomorphogenesis. Secondly, both TAD mutants (mTAD and mWFYL) and DBD mutants (mB and RRQQ) of PIF4 could largely, if not completely, rescue pif4’s hypocotyl growth in the red light (50 µmol m-2 s-1) at 20 °C (Fig. 3j,k; Fig. 5g,h; Fig. 7e,f; Supplementary Fig. 6j,k). Lastly, the mBH variant, which disrupts PIF4 homo- and hetero-oligomerization, failed to rescue pif4’s hypocotyl growth in the red light (50 µmol m-2 s-1) at 20 °C (Fig. 7a,b). Therefore, in the case of photomorphogenesis, PIF4 also plays a key role by collaborating with other PIFs through hetero-oligomerization, while its own DNA-binding and transactivation activities are dispensable.\nThe major conclusion of our manuscript is that functional redundancy has another layer of meaning than the classical understanding. While the latter usually refers to gene redundancy—knocking out a single gene would not lead to any severe consequences thanks to the compensation of related (most likely homologous) genes, our discoveries emphasize that for an essential gene (e.g., PIF4) in a specific pathway (e.g., thermosensory hypocotyl elongation), even if the gene redundancy is minimal—the pif4 single mutant displays a severe defect in thermomorphogenesis, functional redundancy still exists—its DNA-binding and transactivation activity are assisted by other PIFs. That’s why we propose the “functional buffering” model in the Discussion section (lines 561–570). We think this model is well supported by our thermomorphogenetic and photomorphogenetic data. Due to time and space constraints, we decided not to expand our research to PIF4’s other function in this manuscript. We reasoned that at least two prerequisites are needed to conclude PIF4’s molecular glue function in any pathway that it is involved in: 1) PIF4 is indispensable for the pathway—the pif4 null mutant displays a severe phenotype; 2) PIF4 collaborates with other PIFs or cofactors through its hetero-oligomerization domain in regulating this pathway. The essential function of PIF4 in regulating flowering time and stomata development at warm temperatures has been well established4,5; however, whether other PIFs are contributing to these two physiological events at elevated temperatures is yet to be revealed. We are willing to collaborate with researchers who are interested in exploring more in these fields and will make all the related seeds publicly available if requested by our peers.\n3. As argued, phase separation seems not required for PIF4 function in thermomorphogesis. But what is the biological significance of PIF4 condensation? Whether PIF4 interacts with other PIF members to co-condensate in the same droplet?\nResponse: Thank you for this thoughtful question. We, too, were puzzled when we found that PIF4 condensate formation is neither temperature-dependent nor required for thermomorphogenesis. Phase separation is increasingly recognized as an intrinsic property of many proteins, including those with IDRs6, so the in vitro gel-like condensates formed by the PIF4 IDR primarily demonstrate potential rather than physiological necessity. The tobacco assays with full-length PIF4 are closer to the cellular context, yet they still cannot fully recapitulate Arabidopsis, where condensates likely depend on multivalent interactions with endogenous partners (other PIFs, cofactors, and possibly nucleic acids). Ideally, we would monitor condensate dynamics in Arabidopsis lines expressing mGFPmut2-PIF4; however, weak fluorescence in our stable lines prevented reliable in vivo imaging. Nevertheless, our in vitro and in planta assays identify mutations that reduce PIF4’s phase-separation propensity yet do not abolish thermosensory growth, supporting the conclusion that condensation is dispensable for PIF4 function in this context; the biological significance of condensation remains an open question that will require robust imaging in Arabidopsis seedlings to resolve.\nTo address co-condensation, we co-expressed mGFPmut2–PIF4 with mCherry2L-tagged PIF1, PIF3, PIF5, or PIF7 in tobacco. PIF4 co-condensed with each partner in nuclei (Fig. R1). Notably, whereas mGFPmut2–PIF4 formed dozens of nuclear speckles when co-expressed with free mCherry2L, the number of condensates decreased to ≤5 when co-expressed with another PIF tagged with mCherry2L. Because tobacco may not fully recapitulate Arabidopsis macromolecular environments, we chose not to include these preliminary observations in the manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors examined which domains of PIF4 are required for condensate formation and showed that the ability to form condensates is not essential for PIF4’s thermomorphogenetic function. They also tested whether PIF4’s transactivation and DNA-binding activities are required and concluded that they are not. Instead, the authors propose that heterodimerization/oligomerization is necessary. I have a few comments.\n1. Fig. 1b–1d. There are only six circles for 20 °C and four circles for 27 °C. The authors did not describe what these circles represent, but I presume they indicate individual nuclei. Six or four nuclei are too few, especially considering that these are from agro-infiltrated samples. Moreover, agro-infiltrated leaves—and even cells within the same leaf—exhibit substantial variation. The authors need to drastically increase both the number of nuclei analyzed per infiltrated leaf and the number of independent infiltrated leaves. Statistical differences can only be claimed after examining sufficiently large sample sizes. The same concern applies to other figures.\nResponse: Thank you for pointing this out. You are correct: each circle denotes an individual nucleus. We acknowledge that the original sample size was limited; accordingly, we repeated all agro-infiltration assays shown in Fig. 1a–d, 3e, 4g–j, 6g, and Supplementary Fig. 6e. All datasets used for statistical analyses now include at least four leaves (biological replicates), with a minimum of five nuclei per leaf. This information has been added to each figure legend and the Method section (lines 737–738). The new results are consistent with our original data, and the conclusions associated with these figures remain unchanged. We also want to point out that the impact of most mutant variants, especially mWFYL, mDE, mB, and mBH, on PIF4 speckle morphology in tobacco was very obvious and uniform; therefore, the nuclear image showing each PIF4 protein distribution was not cherry-picking but truly representative.\n2. The authors concluded from the FRAP assay (Fig. 1e–1f) that PIF4 forms gel-like condensates in vivo. However, the cytosolic GFP fluorescence signal is also not recovered (Fig. 1e), which calls their conclusion into question.\nResponse: We apologize for the confusion. The FRAP assays shown in Fig. 1e–f were performed exclusively in nuclei. The images displayed GFP fluorescence in representative nuclei, and we did not detect GFP signal outside the nucleus. We have revised the figure legend to state explicitly that FRAP was conducted in nuclei only.\n3. It is difficult to follow how the authors identified TAD1 and TAD2. According to Fig. 1c, m2 (a.a. 7–11) fails to activate transcription, while m16 (a.a. 77–81) retains transcriptional activity. A straightforward interpretation of Fig. S1c suggests four separate TADs (m2, m5–7, m14–15, and m17–18), yet the authors concluded that there are two. Please clarify how the authors arrived at two TADs from these data.\nResponse: Thanks for this thoughtful comment—we agree the link between the mutational data and our TAD nomenclature needed to be clearer. In fact, we also had divided opinions on how to define PIF4 TAD regions within our group.\nThe yeast transactivation assays using three reporters (AUR1-C, HIS3, and LacZ) in Supplementary Fig. 1c indicate four regions whose mutation consistently reduces activation: m2 (7–11 a.a.), m5–m7 (22–36 a.a.), m13–m15 (62–76 a.a.), and m17–m18 (82–91 a.a.). Our initial choice to report two TAD regions followed the precedent for PIF3 by Peter Quail’s group7, where broader regions were defined based on enrichment of activity-impairing substitutions, even though not every residue within those regions contributed, and certain critical residues (e.g., E5) lay outside. By analogy, we designated PIF4 m5–m7 (overlapping APB) as TAD1 and m14–m18 (downstream of APB) as TAD2, leaving m2 unclassified—conceptually similar to the PIF3 E5 case. We included m16 within TAD2 because subsequent analysis showed that a.a. 62–101 (encompassing m14–m18) exhibits a characteristic acidic TAD pattern of alternating acidic and hydrophobic residues (Fig. 4a,b), even though alanine substitutions at m16 did not reduce PIF4 activity in yeast (Supplementary Fig. 1c). Given that most TADs are intrinsically disordered and display length-dependent modularity8,9, our earlier “TAD1/TAD2” labels are best viewed as two interaction interfaces within a broader acidic activation region, rather than fully discrete domains.\nTo avoid ambiguity, we now adopt a purely data-driven nomenclature and label m2, m5–m7, m13–m15, and m17–m18 as TAD1–TAD4, respectively, in the revised Fig. 2a. In the revised manuscript, we clarify that these labels denote four functional subregions contributing to activation in yeast (lines 182–188), and we have updated the text, figures, and figure legends accordingly.\n4. Quail’s group has already identified PIF4 TADs (Dalton et al., 2016), which are virtually identical to those reported here. Thus, it is not accurate to state that “critical features of the PIF4 TAD remain unknown (line 167).” Oddly, the authors cite Dalton et al. (2016) only regarding the lack of interaction of point mutants with phyB. Please revise the relevant paragraphs on PIF4 TAD identification to first acknowledge and cite this prior work.\nResponse: That’s a nice suggestion. We acknowledge that the statement in line 167 was not accurate and didn’t faithfully convey our message. Both Dalton et al. (2016) and Yoo et al. (2021) reported features of PIF4 TAD, mostly by comparing it with the PIF3 TAD based on their homology7,10. We intended to point out that PIF4 TAD had never been systemically mapped and investigated, but the statement failed to deliver that message. We have modified the text to first acknowledge discoveries in both prior publications (lines 168–172).\n5. Although the mTAD version identified in Fig. 2 lost TAD activity in transient assays, it still activated target gene expression in stable transgenic Arabidopsis (Fig. 3). Therefore, the data in Fig. 2 cannot be used to conclude that TAD activity is not required for function.\nResponse: Thank you for the comment, but we respectfully disagree. The modularity and conservation of eukaryotic transcriptional machinery support cross-species assays of activation domains. The yeast transactivation assay has been widely used to test the transactivation activity of transcription factors from other eukaryotic organisms, including plants11. We mapped PIF4 transactivation activity and identified residues whose substitution reduces activity in yeast (Fig. 2b; Supplementary Fig. 1), consistent with prior studies7,10. Transient assays in tobacco further confirmed that the mTAD substitutions diminish PIF4 activation capacity in planta (Fig. 2d–g). Thus, the mTAD allele genuinely impairs the molecular activation function of PIF4.\nDespite this reduction in intrinsic activation, stable transgenic Arabidopsis expressing PIF4 mTAD largely complements pif4 thermomorphogenesis (Fig. 3j–n). A stronger mutant, mWFYL, also complements (Fig. 5g–k). Taken together, these results indicate that PIF4’s own TAD activity is not strictly required for thermomorphogenetic function under our experimental conditions.\nWe acknowledge the apparent paradox. Our data suggest functional compensation in the native context—likely through PIF4’s HLH-mediated oligomerization with other PIFs and/or partnership with other activators (e.g., TCPs)—such that activation domains from partner factors can supply the needed transactivation if PIF4’s TAD is dysfunctional. Consistent with this model, the mTAD version failed to rescue the thermomorphogenetic defects of the pifq (pif1 pif3 pif4 pif5) mutant (Fig. 8b–e). We addressed this point in depth in the Discussion section of the original manuscript, titled “Contribution of the TAD to PIF4-dependent gene expression at warm temperatures” (lines 471-519). We hope this clarifies our reasoning.\n6. The authors showed that mTAD (Fig. 3) and mDE/mWYFL (Fig. 4) form primarily smaller granules both in vitro and in vivo. However, deletion of these regions did not affect granule formation in vitro (Fig. S4). Where does this discrepancy arise? Please clarify.\nResponse: We think this question is related to Question 7. Please see our response below to Question 7 for a detailed explanation.\n7. The authors concluded that “These observations indicate that the TAD functions primarily as a regulator of PIF4 phase separation (line 321).” Since deletion of TAD regions does not affect granule formation (Fig. S4), how was this conclusion drawn? Furthermore, what do the authors mean by “regulator”?\nResponse: We found that specific TAD mutations—mTAD, mDE, and mWFYL—altered PIF4 phase separation in vitro and in vivo (Fig. 3a–i; Fig. 4c–j), whereas deleting the entire TAD did not prevent condensate formation (Supplementary Fig. 4). This pattern is consistent with a regulatory role: the TAD is not strictly required or sufficient for PIF4 condensate assembly on its own, but it modulates the propensity and/or threshold of phase separation in the context of the full-length protein.\nThe discrepant behaviors of full-length proteins with missense mutations and truncated proteins have been commonly observed in proteins with a signal-sensing module or autoinhibitory domain. A good example is the red/far-red light photoreceptor phytochrome B (phyB). Photobody (phyB nuclear body) formation requires the C-terminal output module, and removing the N-terminal photosensory module would not affect phyB phase separation in the nucleus12. However, quite a few mutations in the N-terminal photosensory module can abolish photobody localization of the full-length phyB protein13. Thus, a domain/region can be dispensable for assembly in isolation while still regulating assembly of the intact protein via allosteric or signaling inputs.\nBy “regulator,” we mean that the TAD influences when and how robustly PIF4 forms condensates (e.g., number, size, kinetics), likely by tuning interaction valency or responding to signals, rather than serving as the structural core of the condensate. That said, because we did not provide additional mechanistic evidence to prove this model, we have softened the claim in the revised manuscript and now state that the TAD is insufficient on its own to drive condensate assembly but could potentially modulate PIF4 phase separation (lines 326-328).\n8. mWFYL formed condensates in vitro (Fig. 4c) but not in vivo (Fig. 4g). Similarly, the RRQQ mutation affected condensate formation differently in vitro versus in vivo. These results suggest that the capacity to form condensates differs between in vitro and in vivo conditions. Thus, conclusions should be drawn more cautiously, and the basis for each conclusion should be explicitly stated. For example, the claim that N-terminal regions are non-essential for condensate formation is supported by in vitro but not in vivo data. Shouldn’t the authors temper their conclusions in light of Fig. 4c/4g?\nResponse: Thank you for this important point—we agree that condensate behavior can differ between in vitro and in vivo contexts, and we have clarified the basis of each conclusion accordingly. In vitro assays with recombinant proteins report the intrinsic biochemical propensity of PIF4 to phase separate, whereas tobacco assays reflect that propensity modulated by cellular partners and environments. We believe this, at least partially, explains why mWFYL and RRQQ behave differently across Fig. 4c/Supplementary Fig. 6a (in vitro) and Fig. 4g/Supplementary Fig. 6e (in vivo). Ideally, we would quantify condensates in transgenic Arabidopsis expressing PIF4–mGFPmut2, but weak fluorescence in stable lines prevented reliable imaging; this limitation was discussed in the original manuscript’s Discussion section, “Gel-like PIF4 condensates and their role in thermal responses” (lines 584-610).\nWe also wish to clarify our claims about the “N-terminal regions” and want to let the reviewer know that we didn’t state that “N-terminal regions are non-essential for condensate formation.” If by “N-terminal regions” the reviewer means the TAD specifically (rather than the entire N-terminal IDR preceding the HLH domain), our data indicate that both acidic and hydrophobic residues within the TAD contribute to phase separation: mWFYL and mDE reduce condensate formation, with stronger effects in vivo than in vitro (Fig. 4c,g). If the reviewer means the entire N-terminal IDR, we showed that the extended basic region (C4) is required and sufficient for condensate formation and likely constitutes the core interaction interface, whereas the TAD is neither required nor sufficient, consistent with a modulatory—not structural—role (as discussed in our response to Question 7). These points were stated in the Results sections “Acidic and hydrophobic residues in PIF4 TAD are crucial for its phase separation capacity” (lines 246-276) and “The basic region in PIF4 IDR is essential for its phase separation capacity” (lines 311-353), and further discussed in “Gel-like PIF4 condensates and their role in thermal responses” (lines 584-610).\n9. The authors concluded that “PIF4’s transactivation and DNA-binding activity make only a minor contribution.” However, this interpretation requires more caution. First, as the authors note, PIF4 can heterodimerize/oligomerize with other bHLH proteins. In vivo, PIF4 mutant alleles (mTAD, mB, RRQQ) could dimerize/oligomerize with other PIFs (PIF1/3/4/5/7), thereby binding DNA and activating gene expression. Indeed, the authors showed that mTAD is functional in the pif4 mutant background (Fig. 3) but not in the pif1/3/4/5 background (Fig. 8). A simple explanation is that transactivation activity is supplied by heterodimerized/oligomerized PIFs, which suggests that transactivation activity is in fact necessary. The same reasoning applies to DNA-binding activity. To test this, please also include PIF7, which is known to play an important role in thermomorphogenesis.\nResponse: Thank you for the thoughtful critique. We believe there is a misunderstanding of our conclusion. We did not claim that transactivation or DNA binding are unnecessary for thermomorphogenesis; rather, we concluded that PIF4’s own TAD- and DBD-mediated contributions are minor/dispensable in vivo under our conditions when oligomerization partners are present.\nOur data explicitly support this partner-dependent model: the mTAD allele retains function in the pif4 background (Fig. 3) but fails in the pifq (pif1345) background (Fig. 8). As the reviewer mentioned, these outcomes are most parsimoniously explained by functional compensation via HLH-mediated hetero-oligomerization, whereby partner PIFs (and potentially other activators) supply sufficient transactivation and DNA-binding capacity. In other words, transactivation and DNA binding remain necessary at the network level, but PIF4’s intrinsic TAD/DBD are not strictly required so long as partner factors are available. Conversely, when key partners are removed (as in the pifq background), compensation is lost and the requirement for PIF4’s TAD becomes evident—fully consistent with our interpretation. We realized that our original submission showed only how PIF4 variants affected its homo-oligomerization. To strengthen our argument that PIF4 HLH also mediates its hetero-oligomerization with other PIFs, we performed GST pulldown assays and demonstrated that the mBH variant also dramatically reduced the interaction between PIF4 and some other PIFs (e.g., PIF3 and PIF5), whereas the mB and mTAD versions failed to (Supplementary Fig. 7). We also modified the Results section to emphasize these new data (lines 429-440).\nWhen many readers hear “functional redundancy,” they think of classic gene-level redundancy—loss of one gene is compensated for by other related (most likely homologous) genes. Our case is different. PIF4 itself is essential for thermomorphogenesis (the pif4 null shows a strong defect), but when PIF4 is present, HLH-mediated oligomerization allows it to recruit partner factors (e.g., other PIFs). Those partners’ TADs and DBDs can supply transactivation and DNA binding, making PIF4’s own TAD/DBD dispensable under those conditions, while oligomerization remains critical. In the pifq background, where key partners are absent, this compensation is lost, and PIF4’s TAD becomes required for target gene expression at warm temperatures (Fig. 8). We discuss these distinctions in detail in the original manuscript’s Discussion sections: “Contribution of the TAD to PIF4-dependent gene expression at warm temperatures” (lines 471-519), “Contribution of PIF4’s DBD to its role in thermosensory gene expression” (lines 521-553), and “Contribution of oligomerization to PIF4’s role in thermomorphogenesis” (lines 555-582).\nWe totally agree that PIF7 is important for thermomorphogenesis. Unfortunately, we didn’t have the pif13457 quintuple mutant at hand when generating the transgenic lines. Besides, when we tested whether PIF4 variants would affect its hetero-oligomerization with other PIFs, we could easily detect PIF4’s interaction with PIF3 and PIF5 (Supplementary Fig. 7) but failed to see PIF4-PIF7 interaction using the GST pulldown assay. These results really puzzled us as PIF4 was reported to interact with PIF7 in yeast two-hybrid assays2. However, given that the mTAD variant of PIF4 failed to promote thermosensory hypocotyl growth in pifq (Fig. 8), our argument that PIF4’s TAD function is dispensable while other PIFs are present but is required when its partners are absent is well supported. Therefore, we think the pifq data is sufficient, and it’s not necessary to include PIF7 to strengthen our conclusion.\n10. The authors suggest that PIF4’s HLH domain might possess unique properties that support heterotypic oligomerization with non-PIF cofactors during thermomorphogenesis. However, as I remember, a previously report (Kim et al(?) (2024) Plant Cell) demonstrated that the C-terminal domains of PIF1 and PIF4 are interchangeable, indicating that unique HLH characteristics are unlikely.\nResponse: Thank you for raising this. We agree with the reviewer’s interpretation and have already discussed it in the original manuscript’s Discussion section, “Condensate-independent roles of the PIF4 N-terminal IDR in thermomorphogenesis” (lines 624-635). There, we cite the 2024 Plant Cell paper from Giltsu Choi’s group, which shows that the C-terminal regions of PIF1 and PIF4 are interchangeable14. Consistent with that work, our view is that the HLH module is necessary for dimerization/oligomerization but does not confer unique heterotypic specificity; instead, recruitment of non-PIF cofactors during thermomorphogenesis is more plausibly mediated by features within the PIF4 N-terminal IDR.\nWe think the confusion comes from the last paragraph in one of the Discussion sections, titled “Contribution of oligomerization to PIF4’s role in thermomorphogenesis,” where we listed three potential PIF4-specific features that might determine its central role in thermomorphogenesis. The statement that “PIF4’s HLH might possess unique characteristics that support heterotypic oligomerization with non-PIF cofactors” was the 2nd feature described there. Even though we added a sentence, saying “we will discuss this further in the last section,” we now realize that it created confusion. As mentioned above, our discussion on Choi’s data in a later section basically rejects this possibility. Therefore, we modified these sentences by saying “PIF4 might possess unique structural characteristics that broaden its multivalent interaction capacity with non-PIF cofactors” (lines 578-579).\nReferences\n1. Stavang, J. A. et al. Hormonal regulation of temperature-induced growth in Arabidopsis. Plant J. 60, 589–601 (2009).\n2. Fiorucci, A.-S. et al. PHYTOCHROME INTERACTING FACTOR 7 is important for early responses to elevated temperature in Arabidopsis seedlings. New Phytol. 226, 50–58 (2020).\n3. Lee, S., Zhu, L. & Huq, E. An autoregulatory negative feedback loop controls thermomorphogenesis in Arabidopsis. PLoS Genet. 17, e1009595 (2021).\n4. Kumar, S. V. et al. Transcription factor PIF4 controls the thermosensory activation of flowering. Nature 484, 242–245 (2012).\n5. Lau, O. S. et al. Direct Control of SPEECHLESS by PIF4 in the High-Temperature Response of Stomatal Development. Curr. Biol. 28, 1273–1280.e3 (2018).\n6. Poudyal, M. et al. Intermolecular interactions underlie protein/peptide phase separation irrespective of sequence and structure at crowded milieu. Nat. Commun. 14, 6199 (2023).\n7. Dalton, J. C., Bätz, U., Liu, J., Curie, G. L. & Quail, P. H. A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3. Front. Plant Sci. 7, 881 (2016).\n8. Hope, I. A. & Struhl, K. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell 46, 885–894 (1986).\n9. Hope, I. A., Mahadevan, S. & Struhl, K. Structural and functional characterization of the short acidic transcriptional activation region of yeast GCN4 protein. Nature 333, 635–640 (1988).\n10. Yoo, C. Y. et al. Direct photoresponsive inhibition of a p53-like transcription activation domain in PIF3 by Arabidopsis phytochrome B. Nat. Commun. 12, 1–16 (2021).\n11. Morffy, N. et al. Identification of plant transcriptional activation domains. Nature (2024) doi:10.1038/s41586-024-07707-3.\n12. Qiu, Y. et al. Mechanism of early light signaling by the carboxy-terminal output module of Arabidopsis phytochrome B. Nat. Commun. 8, 1905 (2017).\n13. Chen, M., Schwab, R. & Chory, J. Characterization of the requirements for localization of phytochrome B to nuclear bodies. Proc. Natl. Acad. Sci. U. S. A. 100, 14493–14498 (2003).\n14. Kim, H., Lee, N., Kim, Y. & Choi, G. The phytochrome-interacting factor genes PIF1 and PIF4 are functionally diversified due to divergence of promoters and proteins. Plant Cell (2024) doi:10.1093/plcell/koae110.\nWe thank the reviewers again for allowing us to explain our data further and refine our conclusions. These comments and suggestions have helped us improve our theory and model. In the revised manuscript, we have made our best effort to improve our statements and have implemented changes in the text, figures, figure legends, and supplementary data."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my concerns.", "reviewer_2": "Many of my previous concerns have been addressed properly. However, I think some parts of the manuscript remain misleading and require further clarification. My comments are as follows:\nFRAP assay: In my previous review, I was referring to the nucleoplasmic GFP signal not cytosolic signal. The figures show that both the condensate and the surrounding nucleoplasmic signals recover only marginally. This raises concerns about the authors’ conclusion that PIF4 forms gel-like condensates. The authors should either provide additional data supporting this conclusion or revise it accordingly.\nFigure 3 title and lines 247/248: The current title, “PIF4 TAD is largely dispensable…”, is misleading. Since the authors clarified that this is not their intended meaning—that the TAD is still necessary but may be supplied by other oligomerized PIFs in planta—I recommend revising the description. Alternatively, the authors could insert a phrase directing readers to relevant sections of the manuscript, such as lines 432–435 or the discussion (lines 555–582). Similarly, the authors should review other instances in the main text and supplemental materials where “dispensable” or “not necessary” is used.\nTitle in line 381 and subsequent descriptions: Similar concerns apply here. The current phrasing is highly misleading. As with the TAD, the authors should clarify that DNA-binding activity may be contributed by other PIFs.\nLines 450–454: This section is also highly misleading, or possibly incorrect. As shown in Figure 8e, PIF4 is fully functional in the absence of at least four other PIFs, indicating that hetero-oligomerization is not required for its function. Moreover, mBH lacks both oligomerization and DNA-binding activity, and the function of RRQQ has not been tested in pifQ or pifP. Therefore, the authors cannot claim that the PIF4 dimer is non-functional. Ideally, the authors should test whether PIF4 variants that retain all activities except (hetero-)oligomerization remain functional or not in pifQ or pifP mutants before concluding that a PIF4 (hetero-)oligomer, but not a dimer, is functional. Without such data, the model in Figure 8f should be revised, and all related misleading descriptions in the text should be carefully reworded.\nFigure S7: For “PIF4mB” and “PIF4mBH,” should mB and mBH be formatted as superscripts?"}, "author_response": "Reviewer #1 (Remarks to the Author):\nThe authors have addressed all my concerns.\nResponse: Thank you for your comment.\nReviewer #2 (Remarks to the Author):\nMany of my previous concerns have been addressed properly. However, I think some parts of the manuscript remain misleading and require further clarification. My comments are as follows:\nResponse: Thank you for pointing out these issues. Please see our responses below.\n1. FRAP assay: In my previous review, I was referring to the nucleoplasmic GFP signal not cytosolic signal. The figures show that both the condensate and the surrounding nucleoplasmic signals recover only marginally. This raises concerns about the authors’ conclusion that PIF4 forms gel-like condensates. The authors should either provide additional data supporting this conclusion or revise it accordingly.\nResponse: Thank you for the clarification. We agree with the reviewer. The observation that both the condensates and the surrounding nucleoplasm recover only marginally suggests that nuclear mobility of PIF4 is globally low under our assay conditions. Such FRAP profiles are not, by themselves, diagnostic of a gel-like material state and are equally consistent with binding-dominated or supply-limited regimes (e.g., a small mobile fraction caused by strong chromatin tethering and/or slow nuclear import). Accordingly, we have revised the manuscript to replace “gel-like” with “low-mobility” condensates throughout, and we have added a few sentences to the Discussion section (lines 594–599) to explain this limitation of FRAP and clarify our interpretation.\n2. Figure 3 title and lines 247/248: The current title, “PIF4 TAD is largely dispensable…”, is misleading. Since the authors clarified that this is not their intended meaning—that the TAD is still necessary but may be supplied by other oligomerized PIFs in planta—I recommend revising the description. Alternatively, the authors could insert a phrase directing readers to relevant sections of the manuscript, such as lines 432–435 or the discussion (lines 555–582). Similarly, the authors should review other instances in the main text and supplemental materials where “dispensable” or “not necessary” is used.\nResponse: Thank you for the suggestion; we appreciate the opportunity to clarify our intent. Our use of “dispensable” referred specifically to PIF4’s own TAD in a wild-type background, where partners (e.g., other PIFs) are present and can compensate via high-order oligomerization. In that context, TAD mutations (mTAD, mWFYL) produce only minor effects on thermoresponsive hypocotyl elongation (Fig. 3j–n; Fig. 5g–k). Importantly, we are not suggesting that transactivation is unnecessary for thermomorphogenesis; rather, we conclude that PIF4’s intrinsic TAD can be omitted because transactivation is supplied by oligomerized partners when the network is intact. We summarized the wild-type and TAD mutant PIF4 models in Fig. R1a and R1b, respectively.\nAn analogy may help. Consider a structure supported by multiple pillars: each pillar contributes to stability, but the building remains standing if any single pillar is removed. Under those conditions, one could say an individual pillar is “dispensable” because the design includes redundancy. However, if several pillars are removed and the structure is near failure, removing one more pillar will cause collapse. This mirrors our genetic tests: in pifq, where four key partners are absent, redundancy is largely eliminated, and PIF4’s TAD becomes required for target gene expression at warm temperatures (Fig. 8b–e). By contrast, the wild-type background reflects the natural, redundant network in which the TAD’s contribution can be supplied by partners.\nThat said, we recognize that the word “dispensable” can be misread when stripped of its context (e.g., in a figure title). To avoid ambiguity, we have revised the language to describe the observed effects without the deductive label and to point readers to the partner-dependent model. Specifically, the Fig. 3 title is now: “Loss-of-function mutations in the PIF4 TAD modestly affect thermomorphogenesis.” We have also reviewed the text and Supplementary Information to ensure that any remaining uses of “dispensable” and “not necessary” are tightly qualified by the genetic context or replaced with descriptive phrasing.\n3. Title in line 381 and subsequent descriptions: Similar concerns apply here. The current phrasing is highly misleading. As with the TAD, the authors should clarify that DNA-binding activity may be contributed by other PIFs.\nResponse: Thank you again for the suggestion. Similar to our response to the previous comment, the use of “not required” referred only to PIF4’s intrinsic DNA-binding capacity in a wild-type context with partner PIFs present. We do not imply that DNA binding is unnecessary for thermomorphogenesis overall. Rather, our data support a partner-dependent model in which hetero-oligomerized PIFs supply sufficient DNA-binding activity, allowing PIF4 variants with impaired DBD to retain function in the pif4 background (comparing Fig. R1a and Fig. R1c). From this perspective, our interpretation agrees with the reviewer’s.\nTo improve clarity, we revised the section title and accompanying text. The title now reads “Disrupting PIF4’s DNA-binding ability modestly affects thermomorphogenesis,” a descriptive formulation that reports the result without overinterpretation, allowing readers to assess the implications in context. We also explicitly state that PIF4’s partners, such as other PIFs, may contribute DNA-binding activity (lines 409-411).\n4. Lines 450–454: This section is also highly misleading, or possibly incorrect. As shown in Figure 8e, PIF4 is fully functional in the absence of at least four other PIFs, indicating that hetero-oligomerization is not required for its function. Moreover, mBH lacks both oligomerization and DNA-binding activity, and the function of RRQQ has not been tested in pifQ or pifP. Therefore, the authors cannot claim that the PIF4 dimer is non-functional. Ideally, the authors should test whether PIF4 variants that retain all activities except (hetero-)oligomerization remain functional or not in pifQ or pifP mutants before concluding that a PIF4 (hetero-)oligomer, but not a dimer, is functional. Without such data, the model in Figure 8f should be revised, and all related misleading descriptions in the text should be carefully reworded.\nResponse: Thank you for the comment. Since your points touch several issues, we will address them in turn while keeping our logic explicit.\nFirst, on whether PIF4 is “fully functional” in the absence of four PIFs (PIF1/3/4/5). What counts as “functional” depends on the benchmark. If the term refers to biochemical activity, we agree that PIF4 retains function on its own—in pifq, transgenic PIF4 could still elevate IAA19/IAA29 at 27 °C (original Fig. 8e). That’s also the reason we used the pifq background to test the notion that PIF4’s own TAD would become more important when its partners are absent (please refer to the “pillar” analogy in our response to Comment #2). Please note that PIF4 could still form homo-oligomers in the pifq background, and thus, the gene activation in the pifq background does not necessarily mean that PIF4’s activity is achieved in the form of dimers. On the other hand, if “fully functional” means restoration to wild-type output in Arabidopsis, our data do not support that interpretation. In Fig. 8c, neither PIF4/pifq line (#24 or #4) restores hypocotyl length to Col-0 when transgene expression matches wild type (Fig. 8b). To make this explicit, we repeated the RT-qPCR in Fig. 8e with Col-0 controls. In the revised Fig. 8e, PIF4 could still induce IAA19/IAA29 in the pifq background at 27 °C, but both transcripts remain well below Col-0, consistent with the partial hypocotyl rescue. Thus, PIF4 is active in pifq but not fully functional relative to the wild-type benchmark, indicating that PIF4’s full strength in planta requires partner PIFs.\nSecond, on whether “the PIF4 dimer is non-functional.” We want to clarify that we did not claim that PIF4 dimers are non-functional in the manuscript. Our point is that PIF4’s thermomorphogenetic output relies on its ability to recruit partners. In the first seven figures and supplementary figures, alleles that disrupt PIF4’s own TAD or DBD—mTAD, mWFYL, mB, RRQQ—still largely or fully complement pif4, implying that partner factors can supply DNA binding and/or transactivation when PIF4’s intrinsic modules are compromised (functional redundancy at the complex level). We considered that two candidate features of PIF4—the HLH oligomerization module and the N-terminal IDR—could potentially mediate protein-protein interactions required for partner recruitment. While we haven’t obtained any data to support IDR’s contribution, our mB and mBH data are informative here to support the involvement of the HLH oligomerization domain (Fig. R1a,c,d). If PIF4 functioned strictly as a self-contained dimer without partners, mB (defective basic region) would be unable to bind DNA—either as an mB–mB homodimer or an mB–“other PIF” heterodimer—because bHLH dimers require two functional basic regions to engage DNA1. Yet mB complements pif4 (Fig. 7e–h), arguing that PIF4, in a dimer format, can recruit partners that contribute the necessary DNA-binding capacity. Building on mB, we then disrupted higher-order oligomerization with mBH (basic + HLH). mBH fails to complement pif4 (Fig. 7a–d), and our GST pulldown assays (Supplementary Fig. 7a) show reduced PIF4–PIF interactions, indicating that loss of oligomerization competence abolishes this partner-enabled compensation. We agree with the reviewer that a PIF4 variant that retains all activities except oligomerization will be useful. However, we are not trying to claim that high-order (hetero-)oligomerization is PIF4’s ONLY path to partner recruitment in this manuscript. Additional regions—such as the N-terminal IDR—may also participate, as reflected in our model in Fig. 8f and discussed in the section “Condensate-independent roles of the PIF4 N-terminal IDR in thermomorphogenesis” (lines 630-651). While we are actively testing the IDR’s contribution to PIF4-mediated thermomorphogenesis, our conclusion at present is narrower: oligomerization ability is crucial for PIF4’s thermomorphogenetic function but might not be the only contributor.\nThird, by integrating the full dataset rather than treating each result in isolation, our objective is to define the structural determinants of PIF4’s thermomorphogenetic function. The evidence collectively supports a partner-dependent model in which PIF4 must be present (the pif4 null shows strong defects), but its intrinsic TAD/DBD contributions can be compensated when interaction partners are available. Importantly, these partners are not limited to PIFs; we use the pifq background as a reductionist model to partially remove functional redundancy and show that, in the absence of several key PIFs, PIF4’s own TAD becomes more consequential—when mutated, PIF4 fails to activate thermosensory gene expression. We are not asserting that PIFs are the only partners, nor that the HLH oligomerization module is the sole interface for partner recruitment. It is highly plausible that the N-terminal IDR also mediates compensatory interactions with non-PIF cofactors; indeed, the PIF1–PIF4 domain-swap study from Giltsu Choi’s group suggests a unique role for the PIF4 N-terminus in thermo-induced hypocotyl elongation2. We also mentioned in the Fig. 8f legend that PIF4’s N-terminus is known to interact with multiple cofactors, such as HSFA1d, MED25, HMR, and MRG2. Going forward, we will dissect how PIF4 engages its partners—PIF and non-PIF alike—and test whether specific IDR segments make major contributions to these interactions.\nRecognizing that oligomerization may not be the sole determinant of PIF4 function, we softened the Fig. 8 title to: “PIF4 oligomerization ability is crucial for its thermomorphogenetic function.” We also clarified the Fig. 8 legend: the cross indicates conditions in which PIF4-mediated thermosensory hypocotyl growth is not detectably affected when a given function (intrinsic DNA-binding) is disrupted, whereas the check mark indicates impaired growth under the corresponding disruption (oligomerization).\n5. Figure S7: For “PIF4mB” and “PIF4mBH,” should mB and mBH be formatted as superscripts?\nResponse: Thank you for the suggestion. We confirm that mB and mBH are formatted as superscripts in Supplementary Fig. 7 and its legend, consistent with other variants (mTAD, mWFYL, mDE, RRQQ). During this check, we noticed that Fig. 6 used the labels mKRALL, mKRNLS, mKRBH, and mKRB. To standardize nomenclature and avoid confusion, we have renamed these variants to mALL, mNLS, mBH, and mB, respectively (lines 343–353), and we format them as superscripts when appended to PIF4 (e.g., PIF4mALL, PIF4mNLS, PIF4mBH, and PIF4mB). The text, figures, legends, and supplementary materials have been updated accordingly.\nReferences\n1. Voronova, A. & Baltimore, D. Mutations that disrupt DNA binding and dimer formation in the E47 helix-loop-helix protein map to distinct domains. Proc. Natl. Acad. Sci. U. S. A. 87, 4722–4726 (1990).\n2. Kim, H., Lee, N., Kim, Y. & Choi, G. The phytochrome-interacting factor genes PIF1 and PIF4 are functionally diversified due to divergence of promoters and proteins. Plant Cell (2024) doi:10.1093/plcell/koae110.\nWe thank the reviewer #2 again for allowing us to explain our data further and refine our conclusions. In the revised manuscript, we have made our best effort to improve our statements and have implemented changes in the text, figures, figure legends, and supplementary data."}, {"round": 3, "reviewer_comments": {"reviewer_2": "I think authors still need to edit a few parts to avoid misleading readers (or to avoid overstatement).\n1. Figures 8c–e show that PIF4/pifq is functional, whereas PIF4mTAD/pifq is not. These data clearly indicate that PIF4 can function even in the absence of at least three other PIFs, thus suggesting that PIF4 can work as a homodimer or homooligomer. When functioning as a homodimer/oligomer, PIF4 requires its own transactivation domain (TAD). Similarly, although the authors have not experimentally tested this, it is highly likely that PIF4mB/pifq would also be nonfunctional due to the lack of DNA-binding activity, as homodimeric/homooligomeric PIF4 would require its own DNA-binding domain (DBD).\nI believe the authors also agree with this interpretation, based on the overall logic of the manuscript and their response letter. My concern is that several statements in the manuscript give the impression that PIF4 does not require a TAD or DBD and does not function as a dimer. A few examples of misleading parts are:\n2. Authors retain the original title: “PIF4-mediated thermomorphogenesis relies on its oligomerization ability, not DNA-binding or transactivation activity.” This title gives the impression that DNA binding and transactivation activities of PIF4 are not necessary.\n3. Authors state in the abstract: “By contrast, replacing three conserved basic residues in the first helix of the HLH domain disrupts PIF4 oligomerization and abolishes thermo-induced hypocotyl growth. We conclude that the ability of PIF4 to oligomerize, rather than to bind DNA or recruit the transcriptional machinery, is the primary determinant of its thermomorphogenetic activity.”\nAuthors did not mutate only three amino acids; rather, they mutated both DNA-binding and helix sequences (mBH). To reach this conclusion, the authors would need to show that a PIF4 variant lacking only oligomerization activity is nonfunctional in the pifq background. In addition, although it was not tested, it is likely that PIF4mB/pifq would also be nonfunctional, just as PIF4mTAD/pifq was nonfunctional. Therefore, the authors cannot conclusively state that “the ability of PIF4 to oligomerize, rather than to bind DNA or recruit the transcriptional machinery, is the primary determinant of its thermomorphogenetic activity.”\nBased on the overall logic of the manuscript and the response letter, I think the authors intend to argue that the DBD and TAD are still essential but can be supplied in trans by other PIFs, provided that PIF4 can oligomerize. The same concern applies to several other parts of the manuscript, including the last sentence of the Introduction and the first sentence of the Discussion.\n4. A similar issue applies to the necessity of condensate-forming ability, as the authors have not shown that PIF4mWFYL is functional in the pifq background. I will not elaborate on this point, but the authors may wish to be more cautious here as well, because other oligomerized PIFs could provide intrinsically disordered regions (IDRs) (PIF4mWFYL expressed in wild-type transgenic Arabidopsis could still form condensates in transgenic Arabidopsis because of other oligomerized PIFs’ IDRs).\n5. The model in Fig. 8f depicts a PIF4 dimer bound to target DNA with an “X” mark and PIF4 multimers with a “check” mark. The figure legend states that the “X” mark indicates “is not affected when such a function is disrupted,” whereas the “check” mark indicates “is affected when such a function is disrupted.” Again, this presentation gives the impression that PIF4 does not require its DNA-binding domain and does not function as a dimer.\n6. A similar concern applies to the last sentence of the Results: “Collectively, these data support the notion that the central role of PIF4 in thermomorphogenesis hinges on HLH-mediated hetero-oligomerization with partner factors (e.g., other PIFs), whereas its other canonical TF features, including transactivation and DNA-binding activities, contribute only marginally to thermosensory growth.”\nThe authors cannot conclusively state that thermomorphogenesis “hinges on hetero-oligomerization”, because they did not demonstrate that PIF4 dimers are nonfunctional. Instead, they showed that either PIF4 dimers and/or PIF4 homooligomers are functional in pifq background (Fig. 8e) and PIF4mTAD (presumably PIF4mB) are not functional in pifq background and in that context, canonical features of transcription factor are required."}, "author_response": "Response: Thank you for emphasizing these issues. Please see our responses below.\n1. Figures 8c–e show that PIF4/pifq is functional, whereas PIF4mTAD/pifq is not. These data clearly indicate that PIF4 can function even in the absence of at least three other PIFs, thus suggesting that PIF4 can work as a homodimer or homooligomer. When functioning as a homodimer/oligomer, PIF4 requires its own transactivation domain (TAD). Similarly, although the authors have not experimentally tested this, it is highly likely that PIF4mB/pifq would also be nonfunctional due to the lack of DNA-binding activity, as homodimeric/homooligomeric PIF4 would require its own DNA-binding domain (DBD).\nI believe the authors also agree with this interpretation, based on the overall logic of the manuscript and their response letter. My concern is that several statements in the manuscript give the impression that PIF4 does not require a TAD or DBD and does not function as a dimer. A few examples of misleading parts are:\nResponse: Thank you for the explanation again. We agree with the reviewer’s prediction that PIF4mB would likely be nonfunctional in the pifq background. However, PIF4/pifq versus PIF4mTAD/pifq contrast alone might not imply that PIF4 acts strictly as a homodimer/homooligomer. The pifq background removes PIF1/3/5, but not all potential partners; other PIFs (e.g., PIF7, as the reviewer mentioned in previous revisions) and non-PIF cofactors (e.g., TCP51) could still assemble with PIF4 and contribute TAD/DBD function. Our use of pifq was a reductionist test to show that when partner availability is reduced, PIF4’s own TAD becomes consequential—hence PIF4mTAD is less functional in pifq. On the contrary, in the pif4 single-mutant background, TAD (and probably DBD) mutations do not abrogate PIF4’s function because partner compensation remains available.\nWe appreciate that these findings may feel counterintuitive, especially given the classic view that transcription factors rely on their own DNA-binding and transactivation modules. Our data led us to a partner-dependent model: in a wild-type context, PIF4 variants with disrupted TAD (e.g., mTAD and mWFYL) or DBD (e.g., mB and RRQQ) still support thermo-induced hypocotyl elongation, consistent with compensation from interacting factors when oligomerization is intact. From the reviewer’s comments in this and previous revisions, we believe the reviewer also agrees with this model.\nThat being said, we understand the reviewer’s concern that readers may misinterpret our conclusions. Therefore, we have softened our statements or modified the expression following the reviewer’s suggestions. The detailed changes are pasted in our response to each comment below.\nAs for the oligomerization and dimer function part, we agree with the reviewer that our data has its limitations. We will explain these in detail below, and related changes in the manuscript will be discussed in our response to each comment.\n2. Authors retain the original title: “PIF4-mediated thermomorphogenesis relies on its oligomerization ability, not DNA-binding or transactivation activity.” This title gives the impression that DNA binding and transactivation activities of PIF4 are not necessary.\nResponse: Thank you for pointing this out. We have changed the title to “Oligomerization-competent PIF4 drives thermomorphogenesis through functional redundancy in transactivation and DNA binding.” We believe this title reflects the results more accurately: 1) it emphasizes that the oligomerization function of PIF4 is crucial for thermomorphogenesis without denying the possibility that the dimer form of PIF4 may also contribute, and 2) it implies that PIF4’s partners are contributing to thermo-induced gene expression through their transactivation and DNA-binding activities instead of implying that PIF4’s own transactivation and DNA-binding activities do not play a role in this process.\n3. Authors state in the abstract: “By contrast, replacing three conserved basic residues in the first helix of the HLH domain disrupts PIF4 oligomerization and abolishes thermo-induced hypocotyl growth. We conclude that the ability of PIF4 to oligomerize, rather than to bind DNA or recruit the transcriptional machinery, is the primary determinant of its thermomorphogenetic activity.”\nAuthors did not mutate only three amino acids; rather, they mutated both DNA-binding and helix sequences (mBH). To reach this conclusion, the authors would need to show that a PIF4 variant lacking only oligomerization activity is nonfunctional in the pifq background. In addition, although it was not tested, it is likely that PIF4mB/pifq would also be nonfunctional, just as PIF4mTAD/pifq was nonfunctional. Therefore, the authors cannot conclusively state that “the ability of PIF4 to oligomerize, rather than to bind DNA or recruit the transcriptional machinery, is the primary determinant of its thermomorphogenetic activity.”\nBased on the overall logic of the manuscript and the response letter, I think the authors intend to argue that the DBD and TAD are still essential but can be supplied in trans by other PIFs, provided that PIF4 can oligomerize. The same concern applies to several other parts of the manuscript, including the last sentence of the Introduction and the first sentence of the Discussion.\nResponse: Thank you for the detailed comments. Indeed, these statements in the abstract, introduction, and discussion were not accurate. The reviewer’s interpretation of our central message is sound.\nWe have changed the statement in the abstract (lines 42–47) to “By contrast, substituting twelve basic residues within the basic motif and first helix of the HLH domain, which disrupts both DNA binding and HLH-mediated oligomerization, abolishes thermo-induced hypocotyl growth. These findings support a model in which PIF4’s oligomerization competence contributes significantly to thermomorphogenesis by enabling partner recruitment, allowing DNA-binding and transactivation functions to be supplied in trans.”\nWe have changed the last sentence of the Introduction (lines 123–129) to “By contrast, substituting twelve basic residues, nine in the basic motif and three in the first helix of PIF4’s HLH domain, abolishes higher-order oligomerization and extinguishes thermomorphogenetic growth. We therefore propose a partner-dependent model of PIF4-mediated thermomorphogenetic regulation, in which PIF4’s ability to oligomerize—potentially aided by its long N-terminal IDR—enables recruitment of cofactors that can supply DNA-binding and transactivation in trans.”\nWe have changed the first paragraph of the Discussion (lines 461–476) to “Eukaryotic TFs are traditionally defined by two capacities—sequence-specific DNA recognition and cofactor recruitment, both of which are critical for gene expression modulation. Although PIF4, a canonical bHLH TF, carries the expected machinery for both tasks—an arginine-rich DNA-binding motif and an acidic TAD—our results indicate that in contexts where partner factors are available, PIF4 variants with disrupted DNA-binding or transactivation activity still support thermosensory gene expression and hypocotyl growth, consistent with partner-enabled compensation. Moreover, these two motifs form part of a long N-terminal IDR that promotes PIF4 phase separation in vitro and in planta, but mutations that reduce condensation did not impair PIF4 function under our assay conditions. By contrast, a combined basic-region/first-helix substitution that disrupts both DNA binding and HLH-mediated oligomerization abolishes thermomorphogenetic output, underscoring the importance of oligomerization competence when intrinsic TAD/DBD inputs cannot be supplied by cofactors in trans. We therefore propose that PIF4 drives thermomorphogenesis by assembling multi-protein complexes—likely including PIFs and non-PIF cofactors—such that DNA-binding and transactivation inputs can be provided by partners, while the exact stoichiometry of the active assembly remains to be determined.”\n4. A similar issue applies to the necessity of condensate-forming ability, as the authors have not shown that PIF4mWFYL is functional in the pifq background. I will not elaborate on this point, but the authors may wish to be more cautious here as well, because other oligomerized PIFs could provide intrinsically disordered regions (IDRs) (PIF4mWFYL expressed in wild-type transgenic Arabidopsis could still form condensates in transgenic Arabidopsis because of other oligomerized PIFs’ IDRs).\nResponse: Thank you for raising this point. It is well taken and has been partially addressed in our response to reviewer comment #8 in the 1st revision. We understand the concern as follows: in a wild-type, partner-sufficient context, PIF4mWFYL may still participate in condensates because co-oligomerizing partners (such as other PIFs) contribute their own IDRs, thereby enabling co-condensation in trans. Therefore, it’s not surprising that the mWFYL variant could still complement pif4, as it could still phase separate with co-oligomerizing partners aided by their IDRs. We agree that this is plausible, although we were unable to evaluate PIF4mWFYL condensates directly in Arabidopsis due to weak fluorescence in stable lines (explained in our responses in the 1st revision and mentioned in the revised manuscript).\nOur phase-separation assays—especially the in vitro experiments with recombinant protein—measure PIF4’s intrinsic propensity to form condensates. Our claim is that this intrinsic condensation capacity is not a limiting factor for PIF4’s thermomorphogenetic function under our conditions. We are not suggesting that PIF4 forms condensates only with itself. As with DNA binding and transactivation, condensation can also be supplied in trans by partner factors—such as other PIFs—within mixed assemblies.\nTo incorporate the reviewer’s point, we have revised the Discussion section, “Low-mobility PIF4 condensates and their role in thermal responses.” The last sentence (lines 620–623) now reads: “Thus, in wild-type plants, PIF4’s intrinsic phase-separation propensity is not rate-limiting for thermosensory hypocotyl growth, with condensate features potentially supplied in trans by partner IDRs (e.g., other PIFs and TCPs).”\n5. The model in Fig. 8f depicts a PIF4 dimer bound to target DNA with an “X” mark and PIF4 multimers with a “check” mark. The figure legend states that the “X” mark indicates “is not affected when such a function is disrupted,” whereas the “check” mark indicates “is affected when such a function is disrupted.” Again, this presentation gives the impression that PIF4 does not require its DNA-binding domain and does not function as a dimer.\nResponse: Thank you. We decided to remove all the marks beside PIF4’s structural features. Corresponding descriptions in the Figure 8f legends have also been deleted (after line 1261).\n6. A similar concern applies to the last sentence of the Results: “Collectively, these data support the notion that the central role of PIF4 in thermomorphogenesis hinges on HLH-mediated hetero-oligomerization with partner factors (e.g., other PIFs), whereas its other canonical TF features, including transactivation and DNA-binding activities, contribute only marginally to thermosensory growth.”\nThe authors cannot conclusively state that thermomorphogenesis “hinges on hetero-oligomerization”, because they did not demonstrate that PIF4 dimers are nonfunctional. Instead, they showed that either PIF4 dimers and/or PIF4 homooligomers are functional in pifq background (Fig. 8e) and PIF4mTAD (presumably PIF4mB) are not functional in pifq background and in that context, canonical features of transcription factor are required.\nResponse: Our view is not contradictory to the reviewer’s. As we explained in our response to reviewer comment #4 in the 2nd revision, the interpretation relies on context. We appreciate the reviewer’s focus on the pifq context, but we do not agree that conclusions should be weighted primarily by this background. Our aim is to explain molecular mechanisms and physiological responses in wild-type plants, where partner factors are present. In the wild-type setting where partners are abundant, PIF4 variants with impaired TAD or DBD retain function, whereas eliminating oligomerization competence (mBH) abrogates output. The pifq data are useful precisely because they partially remove redundancy and reveal that PIF4’s intrinsic modules (TAD and presumably DBD) become more consequential; they do not, however, demonstrate that wild-type function relies heavily on homodimers or exclude partner-dependent assemblies.\nAs the reviewer pointed out, the mBH variant abolished more than oligomerization—it also disrupted DNA binding. We noted that in our original submission and thus included mB, which only disrupted DNA binding, as a control. Our comparison of mBH (no rescue) with mB (rescue in pif4) supports the importance of oligomerization competence without asserting that dimers are nonfunctional.\nOperationally, we envision PIF4 recruiting partners as a pre-assembled homodimer or even homotetramer rather than as a monomer. Accordingly, our focus on hetero-oligomerization highlights partner-enabled activity; it does not preclude functional contributions from PIF4 homodimers/homooligomers.\nTo avoid further confusion, we have changed the last sentence of the Results (lines 455–458) to “Collectively, these data support a partner-dependent mechanism in which oligomerization competence is critical for robust thermomorphogenetic output, while PIF4’s transactivation and DNA-binding contributions can be supplied in trans when partners are available.”\nReferences\n1. Han, X. et al. Arabidopsis Transcription Factor TCP5 Controls Plant Thermomorphogenesis by Positively Regulating PIF4 Activity. iScience 15, 611–622 (2019).\nWe thank both reviewers for their constructive suggestions and great support, which helped us improve our manuscript."}, {"round": 4, "reviewer_comments": {"reviewer_2": "The authors have satisfactorily addressed all of my concerns. The revised manuscript now clearly presents the findings and avoids potential misinterpretation through the use of the term ‘supplied in trans.’"}, "author_response": "Response: Thank you again for your wonderful suggestions that helped us revise the manuscript."}]

Decision letter