Variable temperature processing by plasmodesmata regulates robust bud dormancy release

Domain: physics
Published: 2026-01-14
Editorial outcome: major_revision

Abstract
Dormancy is a key mechanism in perennial plants in boreal and temperate regions, protecting buds from winter damage by repressing precocious bud break before spring onset. How plants robustly time dormancy release under fluctuating environments remains unknown. Here, we show that, rather than simply sensing cold duration, buds leverage warm spikes to sense winter progression and time dormancy release. This timing mechanism is mediated by previously unrecognized regulation of plasmodesmata by warm spikes acting through tree ortholog of FLOWERING LOCUS T (FT1) and the gibberellic acid pathway. Our results reveal FT1 as a previously unrecognized, suppressor of callose levels and show that warm spikes repress cold induction of FT1 and GA pathway to suppress PD opening and dormancy release. Importantly, buds exhibit heterogeneity in bud break. This heterogeneity in bud break crucial for bet hedging is amplified under temperature fluctuations and is associated with the thermal responsiveness of plasmodesmata. Altogether, our work reveals dynamic plasmodesmata regulation as a crucial tissue-level mediator of variable temperature processing by buds, enabling robust adaptation of trees to seasonal changes.

Introduction
Boreal and temperate forests represent 45% of the world’s forests1, and experience massive seasonal shifts in temperature throughout the year2,3. To survive these fluctuations, perennial plants such as long-lived trees halt growth and establish dormancy prior to winter4. The growth is reinitiated at the onset of spring, thereby synchronizing growth and dormancy cycles with seasonal changes. Temporal regulation of these cycles is vital for ecosystem function, impacting carbon sequestration5,6, synchronization of tree phenology with that of animals7,8, and forest productivity9.

In autumn, shoot apical meristem (SAM) activity in perennial trees is suppressed, halting leaf primordia formation and growth, and SAM and leaf primordia are enclosed within apical buds10. Following growth cessation, bud dormancy is established11. Dormancy suppresses reactivation of growth in the buds (bud break) until the onset of spring, thereby protecting them from freezing-induced damage during winter12. Premature release of bud dormancy can result in precocious bud break, risking the exposure of buds to extreme low temperatures, causing irreversible damage13–16. Robust temporal regulation of seasonal growth cycles is therefore crucial for tree survival, but perennial trees must also be able to respond to variable climatic parameters.

Prolonged low temperatures (4–8°C) serves as environmental cues sensed by buds to trigger dormancy release upon reaching a chilling threshold17. The antagonistically acting plant hormones abscisic acid (ABA) and gibberellic acid (GA), along with the tree ortholog of the Arabidopsis florigen component FLOWERING LOCUS T (FT1) play a key role in bud dormancy18–25. ABA suppresses cell-to-cell communication by deposition of callose, blocking plasmodesmata (PD) to induce bud dormancy21,25–29. Conversely, low temperatures trigger dormancy release by inducing the expression of FT1, MADS-box transcription factor LIM1 and GA biosynthesis related genes such GA20-oxidase22–24,30.

In nature, daily temperatures can fluctuate between optimal cold interrupted by warm spikes. Consequently, to reliably achieve chilling threshold, buds must distinguish temporary (cold interrupted by long warm spikes) from prolonged (cold interrupted by shorter or a complete absence of warm spikes) cold. While key components of the genetic networks regulating dormancy release have been uncovered4,31, how the buds leverage these to reliably time dormancy release under naturally variable temperature regimes remains enigmatic4,32.

Using hybrid aspen trees, we show that dormant buds do not rely solely on measurement of cold duration but also on the presence and duration of warm spikes to reliably sense the passage of winter and robustly time dormancy release. This mechanism highlighting the previously uncharacterized role of warm spikes, enables buds to distinguish intermittent from prolonged cold and optimally time dormancy release even under variable cold temperatures. This variable temperature processing is mediated by the dynamic response of PD regulated by FT1 and GA. Intriguingly, PD-mediated control of the cell–cell network may also contribute to heterogeneity in bud break crucial for bet-hedging, a survival mechanism for adapting to variable environments, during bud break. This dynamic control of cell–cell communication serves as a previously unrecognized strategy used by trees to adapt and respond effectively to unpredictable environmental changes, thereby enhancing their survival.

Results
Buds process variable temperatures to regulate dormancy release
Trees typically experience cold temperature fluctuations with intermittent warm spikes in nature. It remains unclear how buds process these variable temperature signatures to regulate dormancy release. To address this, we investigated dormancy release by treating hybrid aspen (clone T89) dormant buds to constant cold or simulated temperature fluctuations incorporating low temperatures interspersed with warmer spikes (Fig. 1a). We used bud break following the two temperature treatments of dormant buds to assess dormancy release (as bud break cannot occur until dormancy is released21. The hybrid aspen clone T89 (WT) plants, were exposed to 11 weeks of short days (11WSD) to induce dormancy21 followed by exposure to either constant cold (24 h at 4°C) or variable cold (20 h at 4°C/4 h at 20°C) (Fig. 1a). We compensated for the reduced hours of cold within the variable cold regime by extending the duration of that treatment, so all buds received equivalent hours of cold (Fig. 1a). Following cold exposure, the trees were subjected to warm (22°C) long days (LD; 18 h day/6 h night) to assay bud break. Consistent with earlier results, 100% bud break was observed in dormant buds exposed to 4 weeks of constant cold. In contrast, none of the buds exposed to variable cold underwent bud break, indicating that dormancy had not been released (Fig. 1b, c). Thus, prolonged cold interruption by warm spikes suppresses dormancy release, even if buds receive equivalent cold hours as those exposed to constant cold. This shows that quantitative accumulation of cold hours alone does not account for temperature regulation of dormancy release. Importantly, buds can distinguish between continuous and fluctuating cold, indicating a mechanism for processing variable temperatures to facilitate robust timing of dormancy release in nature.

PD respond dynamically to temperature fluctuations
We next investigated why buds exposed to variable cold failed to break dormancy despite receiving the same total number of cold hours as those exposed to constant cold (Fig. 1a). The blockage of PD by callose is essential for dormancy induction21,26,27. Conversely, low temperature-induced callose reduction, facilitating PD opening, is associated with dormancy release. Therefore, we quantified the callose levels at PD via immunolocalization with callose-specific monoclonal antibody33, in 11WSD dormant buds and after four weeks of either constant or variable cold exposure. In agreement with prior studies, callose levels decreased in dormant buds exposed to constant cold (Fig. 2a, Supplementary Fig. 1a, Supplementary Fig. 2a). However, no such reduction in callose was observed after exposure of buds to variable cold (Fig. 2a, Supplementary Fig. 1a, Supplementary Fig. 2a). Spatial analysis of PD-associated callose levels at a single-cell level showed an increase in the proportion of cells with low callose when buds were exposed to constant cold. This shift was not observed in buds exposed to variable cold (which also failed to trigger dormancy release) (Fig. 2b and Supplementary Fig. 1b–c, Supplementary Fig. 2b). As the abundance and distribution of PD remained unchanged under constant and variable cold (Supplementary Fig. 3), temperature processing appears to act via callose-mediated control of PD opening, not their density. These results showing that PD open in response to constant but not variable cold, point to the crucial role of PD in variable temperature processing in dormancy release.

Constitutively open PD compromise variable temperature processing
As opening of PD is suppressed under variable cold, we investigated whether PD dynamics contributes to temperature processing during dormancy release. To test this, we uncoupled PD opening from its regulation by temperature. If PD gating is important for dormancy release, this should override the inhibitory effect of warm spikes on dormancy release. For testing this, we used a abi1 mutant in which PD are constitutively open. In previous work, we have shown that ABA induces PD closure, and when ABA response is suppressed, as in abi1 mutant, callose levels are highly reduced (Supplementary Fig. 4). Consequently in abi1 mutant PD remain constitutively open21. Importantly, overexpression of PDLP, a PD-specific regulator that reduces trafficking via PD, fully suppresses abi1 dormancy defects (without affecting ABA response due to expression of abi1-1), showing that ABA regulates dormancy through PD closure by inducing callose accumulation in buds21.

We exposed abi1-1 plants and wild-type (WT) control plants to 11WSD, followed by exposure to either constant or variable cold (20 h at 4°C/4 h at 20°C) (Fig. 1a), and then assessed bud break. Both WT and abi1-1 buds broke dormancy under constant cold (Fig. 2c, d), but under variable cold only abi1-1 buds underwent bud break (Fig. 2c, d). Thus, uncoupling PD regulation dynamics from temperature control result in bud break even under unfavorable variable cold conditions. Altogether these data indicate that the regulation of callose-mediated PD opening plays a crucial role in variable temperature processing and robust control of dormancy release.

FT1 mediated control of PD opening is crucial for variable temperature processing
We next investigated upstream regulators of cold regulated dormancy release. Transcription factors SVL and LIM1 have been reported as bud dormancy and PD regulators24,27,34,35. SVL promotes PD closure and positively regulates dormancy, whereas LIM1 acting antagonistically, downregulates callose level to release dormancy. Importantly, dormancy release involves cold mediated downregulation of SVL expression and simultaneously upregulation of LIM1. These results prompted us to investigate their expression in response to variable cold compared with constant cold in buds. Our results show that SVL was similarly downregulated in buds exposed to constant or variable cold (Supplementary Fig. 5a). While LIM1 was upregulated in both constant and variable cold with slightly reduced (statistically not significant) induction in variable cold (Supplementary Fig. 5b). These results showing that SVL/LIM1 expression responds similarly to constant and variable cold suggest that change in SVL/LIM1 expression is unlikely to be the driver of PD-mediated temperature processing.

We then focused on FT1, a cold induced promoter of dormancy release whose loss-of-function impairs dormancy release22,30. To assess FT1’s role in variable temperature processing, we first analyzed its expression in dormant buds under constant and variable cold (Fig. 3a). Upon exposure to constant cold, FT1 expression was upregulated in the hybrid aspen buds, whereas FT1 expression remained significantly low under variable cold conditions. These results suggest that failure to trigger dormancy release under variable cold is independent of transcriptional regulation of SVL or LIM1 but is instead attributable to a failure to induce FT1.

Although FT1 is implicated in dormancy release22, the mechanism by which FT1 contributes to dormancy release remains unknown. Interestingly, our grafting assay (Supplementary Fig. 6), showed that cold treated ft1 buds failed to undergo bud break even when grafted on FT1 overexpressor root stocks (Supplementary Fig. 6). These data strongly hint at PD being closed in ft1 mutant buds. However, in prior analysis of ft1 mutant22, callose levels were not analysed and therefore, it is not known whether FT1 promotes dormancy release by directly acting on PD e.g., facilitating PD opening by downregulating callose. Since FT1 expression remains low under variable cold when callose levels are high and dormancy release is suppressed, we examined FT1’s function in temperature-driven PD regulation and dormancy release.

We first tested if FT1 is essential for cold induced downregulation of callose levels in the buds. For this we used a loss of function ft1 mutant and analysed callose levels in buds before and after cold treatment (Fig. 3b, c) with high-resolution imaging using anti-callose immunogold labeling and electron microscopy. Unlike wild-type buds, where cold triggered callose downregulation, ft1 mutant buds showed no such reduction. Importantly, the PD density in the buds of ft1 mutant was not significantly different between 11WSD and constant cold, as also observed in the wild type (Supplementary Figs. 3 and 7). These results suggest that FT1 is required for PD opening via callose downregulation in response to cold. Moreover, ft1 mutant was not able to undergo dormancy release even after exposure to constant cold (or variable cold with 4h warm spikes) (Fig. 3g and Supplementary Fig. 11a, b). These results highlight the requirement of FT1 for callose downregulation and dormancy release in response to cold.

Next, we investigated if FT1 is sufficient to induce callose downregulation. To test this, we generated transgenic plants in which FT1 could be induced chemically: hybrid aspen plants expressing FT1 were fused with green fluorescent protein (GFP) and hemaglutinin (HA) tags under the control of a steroid (estradiol)-inducible promoter (XVE::FT1-GFP-HA). The XVE::FT1-GFP-HA and WT control plants were exposed to 11WSD to induce bud dormancy. The efficiency of the inducible system was validated by investigating FT1 expression before and after estradiol treatment in the dormant buds, using DMSO (mock control) or 10 µM estradiol (Supplementary Fig. 8), and continuing to keep them under non-inductive short days without exposing them to cold-temperature. Dormant wild-type buds did not show any detectable expression of FT1 before or after treatment, while, in XVE::FT1-GFP-HA plants, FT1 expression was negligible in mock-treated dormant buds but significantly induced after 2 weeks of estradiol treatment (Supplementary Fig. 8).

We then assessed if inducing FT1 expression could reduce PD callose levels using again anti-callose immunogold labeling and electron microscopy. Mock and estradiol treatments had no effect on callose levels in wild-type buds. However, in XVE::FT1-GFP-HA plants, estradiol induced FT1 resulted in significantly reduced callose levels compared to mock-treated buds (Fig. 3d, e). Furthermore, gene expression analysis showed that FT1 regulates the expression of key genes of GA pathway that is known to promote callose downregulation24. FT1 induction promotes the expression of GA20-oxidase, encoding the key enzyme in GA biosynthesis pathway and suppressed the expression of catabolism related GA2-oxidase (Supplementary Fig. 9). Moreover, FT1 induction also modulated the expression of PD-related markers, upregulating the expression of β-1,3-GLUCANASE 1 (BG1) and downregulating PDLP6-2, and MCTP7 expression (Supplementary Fig. 10). These results together with the failure of ft1 mutant to downregulate callose levels in response to cold, highlights the link between FT1 and PD regulation and shows that FT1 upregulation is necessary and sufficient to downregulate callose level.

Since PD opening is crucial for dormancy release (and FT1 facilitates PD opening), we then investigated whether FT1 induction is sufficient to induce dormancy release even in the absence of constant cold temperature using FT1-inducible line. Wild-type and XVE::FT1-GFP-HA plants were exposed to 11WSD to induce bud dormancy. Following this, buds of both wild-type and the inducible lines were exposed to DMSO (mock) or estradiol and assessed for bud break under non-inductive short days without cold treatment (Fig. 3f, Supplementary Fig. 8). Neither mock nor estradiol treatment resulted in bud break in wild-type buds. Similarly, mock-treated XVE::FT1 buds also maintained their dormancy, whereas estradiol-treatment triggered bud break (Fig. 3f, Supplementary Fig. 8). Thus FT1 induction is sufficient and can substitute for cold in inducing PD opening and dormancy release.

These results show that (1) variable cold does not act via SVL or LIM1, (2) FT1 expression responds to different temperature regimes, being induced by constant cold but not by variable cold, and (3) FT1 expression promotes PD opening by reducing callose levels and facilitating dormancy release. Our results thus establish a previously unknown link between the control of PD regulation by FT1. This suggests a scenario where temperature processing is integrated through FT1 expression with variable cold suppressing dormancy release by repressing FT1 induction and antagonizing PD opening.

To validate this further, we generated a double abi1-1/ft1 mutant and tested its response to variable cold. Since PD remain constitutively open in abi1-1 due to reduced ABA response, FT1 should no longer be required for cold-induced PD opening in this mutant. In other words, if temperature processing relies on FT1 to open PD, FT1 should become dispensable once PD are already open. Indeed, our data showed that, in contrast to wild type, abi1-1/ft1 is insensitive to variable cold and undergo bud break (Fig. 3g, Supplementary Fig. 11a–d). Altogether, these results indicate that warm spikes act via FT1 to suppress downregulation of callose which then prevents dormancy release.

Variable cold suppresses GA pathway a regulator of PD
GA (like FT1) is a promoter of dormancy release and has been shown to promote callose reduction at PD in response to cold24,36. For example, hybrid aspen plants overexpressing GA20-oxidase (high GA levels) have low callose and conversely, GA2-oxidase (low GA levels) have high callose levels compared with wild type buds24. Importantly, reducing GA levels, like overexpression of PDLP1, restores dormancy in abi1-1 mutant35 and conversely, exogenous GA application is sufficient to induce callose downregulation and dormancy release23. We first checked if the expression of GA20-oxidase 1, a key enzyme in GA biosynthesis, was, like FT1, differentially regulated by constant or variable cold. Data showed that GA20-oxidase 1 expression mirrored FT1, being upregulated under constant cold but not under variable cold (Fig. 3h). These results suggest that under variable cold, failure to induce GA20-oxidase, may suppress dormancy release (as for FT1). To test this, we used transgenic hybrid aspen plants with elevated GA levels (by constitutive overexpression of GA20-oxidase)37 to uncouple the regulation of the GA pathway from temperature fluctuations. We subjected wild-type and GA20-oxidase overexpressors (GA20oxOE) to 11WSD, followed by constant or variable cold, and then investigated dormancy release (Fig. 3i, Supplementary Fig. 11a, e). Unlike WT, which do not break dormancy, nearly 50% of GA20oxOE plants underwent bud break under variable cold. Thus, uncoupling the GA pathway from temperature regulation compromises buds from processing variable temperature. Altogether, these data indicate that variable temperature processing is mediated by GA together with FT1, two key regulators of PD opening.

Heterogeneity in bud break is enhanced under temperature fluctuations and attenuated in abi1-1 mutant
At a population level, species can mitigate against the impacts of variable and unpredictable environments by generation of heterogeneous responses typically described as bet hedging38. For example, in genetically identical bacterial populations, a small fraction of slow-growing cells enables survival under antibiotics39–41. Bet-hedging has also been reported in plants42–44 but, the underlying mechanisms remain poorly understood.

Since variable responses can protect against unpredictable future conditions, we compared if bud break timing varies between WT buds exposed to both constant and variable cold. We used 2-h warm spikes instead of 4-h warm spikes, as the latter treatment completely blocked dormancy release (Fig. 4a). In contrast with 4-h warm spikes, WT buds were able to undergo bud break when exposed to cold with intermittent 2-h warm spikes (Fig. 4b, Supplementary Fig. 12) and importantly, this corresponded with a significant reduction in PD-associated callose levels in the buds (Supplementary Fig. 13).

Our data showed a substantial variation in bud break timing across populations of genetically identical WT hybrid aspen buds (Fig. 4b). Intriguingly, this heterogeneity in bud break, crucial for bet-hedging, was significantly reduced in abi1-1 trees compared to WT (cov=1.2 in abi1-1 vs. cov=18.72 in WT). Interestingly, variable cold significantly increased the variation in timing of bud break in WT (constant cold cov=18.72 vs. variable cold cov=39.76), indicating a strong enhancement of this response under variable cold compared to constant cold (Fig. 4b, c). In contrast with the wild type, in the abi1-1 mutant, the variation in timing of bud break was reduced compared to WT under both constant (cov=1.2 in abi1-1 vs. cov=18.72 in WT) and variable cold (abi1-1 cov=2.2 vs. WT cov=39.76, suggesting that the mechanism contributing to variability in bud break is compromised in the abi1-1 mutant with open PD. Our results thus show that buds display a heterogeneous response in bud break typically associated with bet hedging. This heterogeneity is enhanced in response to variable temperatures and this response is strongly attenuated in abi1-1 mutant compromised in regulation of PD and variable temperature processing.

Methods
Plant material and growth conditions
Wild-type (WT) hybrid aspen (Populus tremula × tremuloides, clone T89) and transgenic plants were initially grown on half-strength Murashige–Skoog medium (Duchefa) under sterile conditions for four weeks. Following this, they were transferred to soil and grown in a greenhouse with a controlled environment (18-h light/6-hour dark cycle at 22°C during the day and 18°C at night) for five weeks with fertilization. Subsequently, the plants were subjected to short-day (SD) conditions (8-h light/16-h dark cycle at 20°C/15°C) for 11 weeks to induce growth cessation and dormancy21. After 11 weeks under SD conditions, the plants were exposed to either constant cold (24 h at 4°C) for 4 weeks or variable cold (20 h at 4°C/4 h at 20°C) (Fig. 1a). To reflect natural conditions, warm spikes were introduced during mid-day, when warm temperatures are typically encountered. For quantification of the bud break phenotype (Figs. 1c, 2d and Supplementary Fig. 11), all transgenic lines and the wild-type (WT) control were grown and analysed at the same time. For bet-hedging experiment, variable cold was 20h at 4°C and 2h at 20°C (Fig. 4a). The variable cold treatment was extended to ensure that dormant buds in variable cold received an equivalent number of cold hours as buds exposed to constant cold. Following a cold or variable cold, plants were transferred to long-day (LD) (18h light/6h dark) and warm-temperature conditions (20°C) (LD/WT), and bud break was assayed by buds swelling and the emergence of green leaves. For gene expression analysis, apices of WT plants were sampled at different stages: after 11 weeks of SD treatment (11WSD), and after two weeks (2WC) and four weeks (4WC) of exposure to the constant or alternating cold/warm conditions. As indicated above, for variable cold, bud samples were collected so that buds had received an equivalent cold hours at 2 or 4 weeks as constant cold-treated buds at these time points. Each collected sample was immediately frozen in liquid nitrogen and stored at −80°C for future analysis. Photographs of the apices were captured using a Canon EOS digital camera to document the bud break process.

RNA isolation and qRT-PCR analysis
Total RNA was extracted from plant shoot apices using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich). RNA (10μg) was treated with RNase-free DNaseI (Life Technologies, Ambion) to remove contaminating DNA. Subsequently, 1μg of this RNA was used for cDNA synthesis using the iScript cDNA Synthesis Kit (BioRad). Ubiquitin was used as the reference gene in all experiments24. Quantitative reverse transcription PCR (qRT-PCR) was conducted using a LightCycler 480 SYBR Green I Master mix and a LightCycler 480 II instrument (both from Roche). The relative expression levels of target genes were calculated using the Δ-cq method, as previously described68. Details of the primer sequences used for qPCR are provided in Table S1.

Generation of transgenic lines
To generate abi1-1/ft1 transgenic lines, FT1-pHSE401 (FT1-Crispr)24 construct was transformed in abi1-1 background via Agrobacterium-mediated transformation21. Generation of GA20oxOE transgenic line has been described previously (36). To generate inducible XVE::FT1-GFP-HA line, FT1-GFP fragment69 was amplified using the primers listed in Table S1. The amplified fragment was cloned into pENTR/D-TOPO (Invitrogen) and subsequently transferred into the plant transformation vector pDMC7. Hybrid aspen transformation was performed as described previously21.

Estradiol treatment
Estradiol treatment was performed by gently removing the bud scales of plants that had undergone growth cessation and dormancy establishment. The exposed buds were then dipped in either 10 µM estradiol or DMSO solution. To prevent wilting, the plants were covered with a transparent bag. The plants remained in short-day conditions and were treated with estradiol every third day. For gene expression and PD analysis, the treated bud samples were collected after 2 weeks. For gene expression analysis, samples after collection were immediately frozen in liquid nitrogen. For bud break analysis, swelling and emergence of green leaves were monitored.

Grafting experiment
FT1ox and ft1 Crispr plants were grown in chambers under short photoperiod conditions (SD) (8h, 20°C light/16h, 15°C dark cycles, 80% relative humidity) for 11 weeks and transferred to cold for 5 weeks. Scions of the ft1 plants were then grafted onto rootstocks of ft1 Crispr and FT1-overexpressing plants, as described previously21. Grafted plants were kept under LD and monitored for bud break.

Transmission electron microscopy (TEM) sample preparation, imaging, and PD mapping
Samples for TEM were prepared as described earlier21. Buds from 11 weeks of short days and 4 weeks of constant and variable cold temperature after removing bud scales and outer leaf primordia were fixed at room temperature (RT) in a slowly agitating shaker overnight in a solution containing 2% paraformaldehyde, 2% glutaraldehyde in 0.05M cacodylate buffer, and 10mg/ml tannic acid. Samples were then rinsed in cacodylate buffer (0.05M) and post-fixed in 1% osmium tetroxide (in water) for 1 h at RT in darkness. Following post-fixation, samples were dehydrated through a graded ethanol series (30–100%) and propylene oxide (100%), then embedded in Spurr resin (S031/D) in casting molds and polymerized at 70°C for 24 h.

The fixed buds embedded in Spurr blocks were longitudinally sectioned into 90nm thick slices using an EM UC7 ultramicrotome (Leica) and placed onto 100 mesh copper grids. Observations were carried out using a FEI TECNAI Spirit 120kV electron microscope. To acquire data for PD mapping, high-resolution individual TEM images (at a magnification of 4400x) were captured and stitched together using the ImageJ software to reconstruct the entire meristem surface (Supplementary Fig. 14). PD were identified and counted manually in approximately 50 cells. The Inkscape software was then used to create a color map based on the acquired data, reflecting the number of PD at cell-to-cell interfaces (Supplementary Fig. 14).

Callose immunostaining
The fixed buds embedded in Spurr blocks were longitudinally sectioned using an EM UC7 ultramicrotome (Leica). For immunofluorescence, 200nm thick slices were placed onto poly-lysine-coated slides. For immunogold, 90nm thick slices were placed onto 100 mesh copper grids. Both techniques followed the same steps up to the primary antibody incubation, with the differences starting from the secondary antibody step onward.

Right after sectioning, immunolabelling was performed using a modified version of a previously described protocol70. All solutions were prepared in PHEM buffer, comprising 60mM PIPES, 25mM HEPES, 10mM EGTA, and 4mM MgSO4·7H2O. To prevent nonspecific binding, the samples were subjected to a blocking step using 5% neutral donkey serum (NDS) before incubation with the primary antibody. The callose antibody (obtained from Australia Biosupplies) was appropriately diluted to 1/20 in PHEM buffer supplemented with 5% (v/v) NDS and incubated with the samples at room temperature for 2 h.

For immunofluorescence, after washing with PHEM buffer (5 times for 5 min each), the secondary anti-mouse antibody, coupled to Alexa Fluor 555, was diluted to 1/100 in PHEM buffer containing 5% (v/v) NDS. This antibody solution was then applied to the samples and incubated for 2 h. Following another series of washes in PHEM buffer (5 times for 5 min each), 1mg/ml of calcofluor was applied for 10 min to stain the cell walls. Finally, the slides were rinsed with Milli-Q water.

For immunogold labeling, after washing with PHEM buffer (5 times for 5 min each), the secondary anti-mouse antibody, coupled to 10nm EM gold particles, was diluted to 1/30 in PHEM buffer containing 5% (v/v) NDS. This antibody solution was then applied to the samples and incubated for 2 h. Following another series of washes in PHEM buffer (5 times for 5 min each), the samples were finally rinsed with Milli-Q water.

TEM imaging and callose quantification
Immunogold-labeled sections were imaged using a FEI TECNAI Spirit 120kV electron microscope. High-resolution individual TEM images (at a magnification of 42,000x) were captured to quantify callose deposition by counting the number of gold particles. For normalization, a rectangular region of 0.025 μm² was delineated around the plasmodesmata. This quantification was performed across different genotypes and experimental conditions.

Confocal microscopy
All confocal imaging experiments were conducted on a Zeiss LSM880 microscope, operated with ZEN Black 2011 software. The slides, obtained from immunofluorescence, were mounted with a thin layer of Milli-Q water and a coverslip, and images were captured using a Plan-Apochromat 40x oil objective. Alexa Fluor 555 fluorescence was detected using an Ex 561nm and Em 561–615nm filter, while calcofluor fluorescence was detected using an Ex 405nm and Em 411–476nm filter.

Data extraction, analysis and display
Confocal microscopy stacks with two channels, one labeling cell walls and the other staining PD with callose, were used (Supplementary Fig. 15A). A custom ImageJ toolset was employed for the automated segmentation of cells, walls, and PDs while preserving relational information (Supplementary Fig. 15B–C). The toolset includes tools for CZI to Zip conversion, single file analysis, multiple file analysis, and data compression.

The analysis involved several key steps. The original wall image was pre-processed to enhance and skeletonize the walls. Tri-junction points (points where three walls converge) were identified and used to segment the skeleton into individual wall segments. Cells were then identified based on the areas enclosed by walls. For PD, the original image was pre-processed, and local maxima were detected. The toolset then extracted relational data: Each PD was associated with a wall, each wall segment with two adjacent cells, and each junction point with three connected walls.

The toolset outputs composite images with multiple channels allowing visual inspection of detections, CSV files containing data on cells, junction points, PDs, and walls, a comprehensive JSON file containing all extracted data, analysis parameters, and image metadata, and PNG image files of each channel. The toolset exposes parameters for preprocessing, detection, and parent search tolerances, allowing users to adjust the analysis according to specific needs.

The toolset, example datasets, and example output are available from the following GitHub: https://github.com/fabricecordelieres/IJ-Toolset_PD-analysis/tree/main.

Further analysis and data visualization are made possible using a Python script. Briefly, the script generates several visual outputs to present PD density at several granularities (per wall, per length of wall, per cell). It also outputs numerical data in the form of table data to ease further statistical analysis. For each cell-cell interface, the wall segment length is exported, along with the number of PD and their total intensity, allowing to extract their density. The Python code, example datasets, and example output are available from the following repository: https://gitub.u-bordeaux.fr/gmaucort/plasmodesmata_import_display_report.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This research group recently published a lot of important findings about tree bud dormancy using genetic approaches. Especially, they reported that ABA has central role for bud dormancy establishment and proposed the dense TEM signal in plasmodesmata (they called plasmodesmata sphincter, Tylewicz et al., 2018, Science) is found only in normal WT but not in abi1-1 mutant, suggesting it is critical morphological marker of dormancy establishment. They also reported that low-temperature-induced dormancy release (later stage) is mediated by LIM1, FT1 and GA pathways (Pandey et al., 2024, EMBO J.). These published studies are consistent with important pioneer findings published by van der Schoot group [Rinne et al. (2001), Rinne et al. (2011)], which have facilitated recent bud dormancy study. This study demonstrated that warm spikes during dormancy release period suppress bud break through the thermal responsiveness of plasmodesmata. Moreover, GA pathways are also involved in this response. Quantification of callose levels in plasmodesmata using callose-specific antibody and genetic approach using transgenic plants provided solid evidence to support some parts of their thoughts. My first concern is the appropriateness of the use of the word “robust bud dormancy release” in the title. As previously reported, dormancy sphincter removal should be the right word of “dormancy release”, to my understanding. Therefore, the process from plasmodesmata closure to opening may represent “later stage of dormancy release” or “the stage towards bud break”. The topic in this study is not about dormancy sphincter removal but PD opening, which corresponds to “late dormancy release towards bud break”. Therefore, I suggest the use of “bud break” rather than “dormancy release” for the title. Fig5 (a) model does not represent “dormancy release” but “bud break transition”. My second concern is the link between bet hedging (heterogeneity in bud break) and plasmodesmata. Although the authors proposed there is close link between them in the abstract and Fig. 4, the authors did not investigate callose level in buds under variable cold (2h-W). They also did not investigate callose level in abi1-1 mutant at all. Under this circumstance, the relationship between plasmodesmata and bud break heterogeneity or bet-hedging has not been well-validated in this study. Fig4 title is not appropriate. ABA controls PD sphincter (Tylewicz et al., 2018) but ABA also important for callose level in PD? Is this validated? There are other comments 1. While FT1 and GA20OX show similar expression patterns under the 11WSD, Cold, and Variable Cold treatments, this observation alone may not be sufficient to clarify how FT1 regulates PD opening independently of the LIM1 and SVP pathways. Additional mechanistic evidence would help substantiate the proposed model. 2. In Fig. 3. Including ft1 knockout comparisons under both Cold and Variable Cold conditions would substantially strengthen the conclusions. This would clarify whether FT1 is essential for temperature-induced dormancy release and whether its reduced expression under Variable Cold is a primary cause of dormancy failure. Without this analysis, the proposed role of FT1 remains speculative. 3. The authors present an interesting and valuable set of phenotypic data for abi1-1, (ft1, should be included) and the abi1/ft1 double mutant under various environmental treatments. However, the underlying regulatory mechanisms remain unclear. To better understand the genetic interactions and clarify whether ABI1 and FT1 act in the same or parallel pathways, it would be helpful to analyze the expression levels of key genes such as SVL, LIM1, FT1, and GA20ox in these mutant backgrounds. Specifically, comparing gene expressions across 11WSD, constant cold, and variable cold conditions could provide more direct evidence of the regulatory roles and relationships among these genes. This would significantly strengthen the mechanistic interpretation of the observed phenotypes under variable cold. Fig. 1b, Fig. S7- Could the authors clarify why the WT plant in Fig. 1b (11WSD) appears to retain its leaves, while the WT plant shown in Fig. S7 (10WSD) seems to have entered dormancy and shed its leaves? This apparent inconsistency raises some confusion—was there a mistake in photo selection or labeling? Or is there another explanation?", "reviewer_2": "Pandey et al. address how poplar buds are released from dormancy by environmental signals, thus adding to an existing body of work on this highly important physiological response of plants and the end of the winter season. The authors describe and investigate a previously insufficiently characterised mechanism, whereby poplar plants suppress bud break in response to fluctuating outside temperature. Understanding plant responses to such \"real life\" scenarios and variable environments is highly relevant, not only for our fundamental understanding of plant development, but also to explore and possibly predict plant responses to changing current and future climate scenarios. The authors investigate the expression and functional role of key regulators of bud break in poplar, including transcription factors, hormones, and mobile factors that are known as floral inducers, such as FT1. The manuscript reports on a key role for FT1 in triggering dormancy release, mediated by opening of plasmodesmata by reducing callose levels, and how short warm periods that interrupt cold phases can basically erase the effect of extended cold, resulting in suppression of bud break. The authors propose that FT1 is a key factor in this response, and that the underlying mechanism supports bet hedging, i.e. a differential response of buds to the same environmental input in order to increase reproductive success. The experiments are well presented and described. I very much enjoyed the high quality of the analysis of callose deposition at PDs, based on antibody detection using fluorescence labelling for entire meristems, and detailed surveys via electron microscopy with gold labelled antibodies! Overall, a well written manuscript which addresses dormancy and the role of plasmodesmata in bud break from a new angle, which was a pleasure to read. Nevertheless, there are a few points that the authors should consider and address to further strengthen the manuscript, which I have detailed below. 1) A key point the authors make in the manuscript is that FT1 expression is sufficient and can subsitute for cold in inducing PD opening and dormancy release. This statement is based, among other experiments, on an inducible FT1 transgenic line, which can overcome environmental regulation and induce bud break and PD opening (judged by reduction in callose). However, the authors have analysed changes in callose accumulation at PDs several weeks after FT1 induction, so FT1 might be a rather indirect regulator of PD callose. This problem (correlation, causality) could be resolved by assaying PD callose levels shortly after FT1 induction, and before bud break. In that context, I would like to quote a sentence from the manuscript, which highlights this more: \"Altogether, these results indicate that warm spikes act via FT1 to suppress downregulation of callose which then prevents dormancy release\". It would be equally possible to argue that warm spikes act via FT1 (and possible LIM1) to prevent dormancy release, which would normally result on PD opening and callose reduction. 2) To determine whether defects in callose deposition/degradation could affect bud dormancy break the authors used the abi1 mutant, which was previously reported to break dormancy even in cold condition, because plasmodesmata are always open in this mutant background. They showed here that the mutant displayed dormancy break also in the variable cold condition, i.e. with warm spikes interrupting the cold period.; also, they are insensitive to the warm spikes, that inhibit dormancy break in wt condition. Determining the level of callose at PDs in the three conditions (11WSD, constant cold and variable cold) in the abi1 background is important to evaluate whether a decrease in callose accumulation at the plasmodesmata is necessary and sufficient to trigger break dormancy. 3) One feature to assess bud dormancy is the presence of lipid droplets. As a matter of fact, the authors nicely observed lipid droplets in the 11WSD treatment. The information whether also buds from variable cold condition showed lipid droplets is missing (lipid droplets of what??) 4) A reported pathway to trigger dormancy release relies upon the simultaneous downregulation and upregulation of SVL and LIM1, respectively. The authors therefore assayed expression of SVL and LIM1 in entire buds by qRT-PCR in the three different conditions. They point out that under both continuous cold and warm spiked conditions, SVL is still repressed, and LIM1 is still induced. FigS4 shows, in my opinion, a strong difference in the response of LIM1 under the two key conditions. Is that statistically different? Do all cells in a bud behave in the same manner, i.e., could there be regional differences in expression levels? A possible interpretation of the data, which differs from the authors point of view, could be that LIM1 is not sufficiently induced in the warm-spiked situation, and therefore dormancy is not broken. 5) FT1 has been previously reported to be a cold induced promoter of dormancy release. To determine whether inhibition of dormancy release in variable cold condition was due to a failure in triggering FT1 expression, the authors performed qRT-PCR on entire buds, testing FT1 expression in the three conditions. They observed a decrease in FT1 expression in variable cold condition compared to the constant cold, suggesting that the failed dormancy release in variable cold condition was caused by reduced levels of FT1 expression in the buds. Statistical analysis would strengthen this dataset; however, given that LIM1 expression levels were also affected in variable cold conditions (compared to constant cold), focussing only on FT1 now seems to ignore other possible pathways and scenarios. 6) The authors showed here that the ft1 mutant buds do not show a decrease in callose (associated to the number of gold particles) in cold condition. Different callose levels could also be caused by a different number of PDs. Analyzing the number of plasmodesmata in ft1 mutant would be important to determine how FT1 act in dormancy release. Furthermore, the data presented here are in contrast to data presented by André et al., 2022, which uncouple FT1 function and callose presence at PDs in poplar (DOI: 10.1016/j.cub.2022.05.023). This should be discussed. 7) The authors showed that the double mutant abi1-1/ft1 presented bud break even in variable cold condition. They concluded that warm spikes act via FT1 to suppress downregulation of callose to prevent bud break. I think that their conclusions are not supported by the data and additional observations are needed. Murata et al., (2025) very recently reported that cold triggers ABA signaling that in turn induces callose synthesis by upregulation of CALLOSE SYNTHASE genes, restricting FT translocation to the SAM in Arabidopsis thaliana. The authors here suggested a direct activity of FT1 on degradation/deposition of callose, modulating dormancy break. Analysis of FT1 translocation in abi1-1 mutant would be important to address whether movement of FT1 in the bud is important for dormancy break or FT1 solely act in modulating callose distribution at PDs. Do expression patterns of FT1, and local decrease of callose overlap, or does this occur in different, separate cells? I.e. is FT1 transport required for the observed effects? 8) Based on their results, the authors concluded that FT1 (together with GA) trigger dormancy break but their relationship is not further investigated. Enhanced expression of FT1 could be the effect and a consequence of callose degradation/reduced callose deposition at PDs and not the trigger of the response. Analysis of FT1 expression in the GA overexpression lines and analysis of dormancy release in GA20oxoOE ft1 doble mutant would clarify this aspect. 9) The authors here reported that level of expression of FT1 are lower in variable cold condition compared to cold condition; considering that the ft1 mutant presented defects in dormancy break even in cold condition they concluded that warm spikes inhibit bud break. FT1 expression was quantified by qRT-PCR on the entire bud. Analysis of the pattern of expression of FT1 would determine whether the accumulation of callose is inhibited in the same cells where FT1 expression is triggered or if FT1 acts non-cell autonomously to determine dormancy break. 10) LIM1 regulation is one of the key responses to cold, and upregulation is reduced in variable cold conditions (Fig S4). could you provide some statistical analysis of these data? Are these qRT-PCR data? How would LIM1 mutants respond to variable cold, i.e., is LIM1 here the key factor that mediates the difference in response? 11) The suggested bet-hedging strategy is certainly intriguing, but not really explored sufficiently in this manuscript. As stated above (point 9), I am missing the spatial component in the analysis of bud break. Are the breaking/non-breaking buds evenly distributed at the plant? How is the response of a bud, consisting of many hundred of cells, overall integrated? I am tempted to suggest to take this part of the story out of the manuscript, and explore this in-depth in a separate manuscript. It appears here like a very interesting, but insufficiently supported add-on.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "This study provides important mechanistic insight into how plants integrate variable temperature signals to regulate bud dormancy release. The authors demonstrate that plasmodesmata-mediated cell–cell communication acts as a key integrator of fluctuating thermal cues, particularly through the detection and interpretation of intermittent warm spikes. They show that long warm periods suppress dormancy release by preventing PD reopening via repression of FT1 and GA20-oxidase expression. In contrast, shorter warm interruptions allow cold integration and promote dormancy release. These findings reveal a previously uncharacterized role for FT1 in callose downregulation and PD opening. The study also links temperature-responsive PD dynamics to phenotypic variability in bud break, suggesting a cellular basis for bet-hedging under unpredictable spring conditions. Overall, this work presents a compelling and original framework for understanding how plants maintain robust developmental timing amid environmental noise, with broader relevance to plant adaptation under climate change. The manuscript is well written, and the data presented are convincing, novel, and well suited for publication in Nature Communications. Below are a few comments and questions for the authors' consideration: Comments: Figure 1a and Figure 4a The schematic suggests that warm spikes were applied at different times on consecutive days (e.g., from ZT20–ZT24 on the first day, and from ZT40–ZT44 on the second). Is this interpretation correct? If so, could the authors clarify the rationale for this design? Figure 2, Figures S1 and S3 and discussion The observation of stronger callose deposition at PDs in the SAM compared to adjacent tissues raises an interesting possibility of cold-induced, cell–cell communication regulation. Do FT1 or GA biosynthesis genes exhibit SAM-specific expression patterns under these conditions? Could this reflect SAM-specific expression of β-1,3-glucanases involved in PD opening? Line 119 “In agreement with prior studies, callose levels decreased in dormant buds exposed constant cold (Fig. 2a, Fig. S1a, Fig. S2a).” Could the authors clarify how the measured callose levels were specifically linked to PD openness? This connection is not immediately evident from the provided images. Line 238 The data show that constitutive overexpression of GA20ox leads to bud break in nearly 50% of plants under variable cold conditions (Fig. S6). However, this phenotype does not fully phenocopy the effects of FT1 induction. Could the authors elaborate on potential reasons for this divergence? Does this suggest that FT1 may regulate additional GA-independent pathways required for PD opening and dormancy release? Line 292-297 if cold integration mechanism that enables robust sensing of autumn to late autumn/winter transition, as, typically, there is a shift towards progressively shorter periods of warm spikes relative to cold. A lack of response to cold when the warm spikes last longer can therefore ensure that cold registration is not initiated and chilling threshold is not achieved prematurely, preventing precocious dormancy release….. (Fig.5) Is callose deposition at PDs, and the resulting closure of cell–cell communication, only triggered when warm spikes become short enough? If so, at what point in this model does PD dynamics shift from closure to opening?\""}, "author_response": "Dear Editor, Please find enclosed our detailed response to reviewers. We have addressed the reviewers’ comments point by point experimentally and adding new results to the revised manuscript as well as via additional clarifications and revised the manuscript accordingly. We hope our response with the revisions will be satisfactory for acceptance of our manuscript for publication. Sincerely, Rishikesh Bhalerao Response to reviewers: Reviewer #1 (Remarks to the Author): This research group recently published a lot of important findings about tree bud dormancy using genetic approaches. Especially, they reported that ABA has central role for bud dormancy establishment and proposed the dense TEM signal in plasmodesmata (they called plasmodesmata sphincter, Tylewicz et al., 2018, Science) is found only in normal WT but not in abi1-1 mutant, suggesting it is critical morphological marker of dormancy establishment. They also reported that low-temperature-induced dormancy release (later stage) is mediated by LIM1, FT1 and GA pathways (Pandey et al., 2024, EMBO J.). These published studies are consistent with important pioneer findings published by van der Schoot group [Rinne et al. (2001), Rinne et al. (2011)], which have facilitated recent bud dormancy study. This study demonstrated that warm spikes during dormancy release period suppress bud break through the thermal responsiveness of plasmodesmata. Moreover, GA pathways are also involved in this response. Quantification of callose levels in plasmodesmata using callose-specific antibody and genetic approach using transgenic plants provided solid evidence to support some parts of their thoughts. My first concern is the appropriateness of the use of the word “robust bud dormancy release” in the title. As previously reported, dormancy sphincter removal should be the right word of “dormancy release”, to my understanding. Therefore, the process from plasmodesmata closure to opening may represent “later stage of dormancy release” or “the stage towards bud break”. The topic in this study is not about dormancy sphincter removal but PD opening, which corresponds to “late dormancy release towards bud break”. Therefore, I suggest the use of “bud break” rather than “dormancy release” for the title. Fig5 (a) model does not represent “dormancy release” but “bud break transition”. Response: We thank the reviewer for raising an important point. We are happy to revise the title to include the reviewer’s point of view to: Variable Temperature Processing by Plasmodesmata Regulates robust Dormancy release preceding bud break if necessary. However we would like to include a clarification regarding the relationship between analysis of dormancy sphincters and callose related to PD and dormancy. In buds, bud break can only proceed if dormancy is released. Crucially, in context of dormancy regulation, PD closure is essential for dormancy establishment whereas their opening is essential for dormancy release as shown by prior studies by Rinne et al and others. Accordingly, Rinne and others (including us) have interchangeably used either callose quantification (using anti-callose antibodies) or “dormancy sphincters” (that are visualized by TEM as electron dense structures) as a marker for PD closure/opening associated with dormancy establishment or its release. Ours as well as results of others show that analysis of dormancy sphincters or callose quantification basically give same results. For example, in the wild type at 11 WSD both callose levels and dormancy sphincters are high whereas after cold treatment at both dormancy sphincters and callose levels are low. Similarly, in abi1-1 we have previously shown that dormancy sphincters are very low at 11 WSD and we show here (see below Fig. 2) that callose levels are also very low, consistent with the open PD phenotype. Thus, the status of closure or openness of PD associated with dormancy regulation can be measured either with loss of dormancy sphincters or alternatively by callose measurements. Here, we chose quantification of callose levels (although they are far more challenging to do) than looking at dormancy sphincters since callose quantification provides a better cellular resolution for PD opening (as a prerequisite for dormancy release). Based on this clarification we would appreciate if the reviewer would consider to agree with us to retain original title. If not we can change the title as indicated. My second concern is the link between bet hedging (heterogeneity in bud break) and plasmodesmata. Although the authors proposed there is close link between them in the abstract and Fig. 4, the authors did not investigate callose level in buds under variable cold (2h-W). They also did not investigate callose level in abi1-1 mutant at all. Under this circumstance, the relationship between plasmodesmata and bud break heterogeneity or bet-hedging has not been well-validated in this study. Fig 4 title is not appropriate. ABA controls PD sphincter (Tylewicz et al., 2018) but ABA also important for callose level in PD? Is this validated? Response: We thank the reviewer, and to address this concern, we have now provided two additional evidences as requested. First, we measured callose levels under 2 hour warm spikes. Our data shows that in contrast with 4 hour spikes, 2 hour spikes are able to significantly reduce callose levels, and this reduction correlates with dormancy release (this data has been added as sup Fig S13). As a response to reviewer’s suggestion, we have also changed the title of Fig. 4 to: The bet-hedging response of buds is altered in abi1-1 mutant Second, regarding the abi1-1 mutant, we now present a new data (see below) showing that after 11 weeks under short-day conditions, callose levels are already significantly lower in the abi1-1 mutant than in wild-type plants, consistent with our previous findings based on analysis of “dormancy sphincters” (Tylewicz et al., 2018). We hope this addresses reviewers’ concern. This data has also been added to revised manuscript (as supp Fig S4). There are other comments 1. While FT1 and GA20OX show similar expression patterns under the 11WSD, Cold, and Variable Cold treatments, this observation alone may not be sufficient to clarify how FT1 regulates PD opening independently of the LIM1 and SVP pathways. Additional mechanistic evidence would help substantiate the proposed model. Response: We have shown earlier that FT1 redundantly of LIM1 regulates GA pathway that is promoter of PD opening (Pandey et al., 2024). However to further address reviewers’ comment, we have now added data from inducible FT1 line (XVE:FT1) to show that FT1 induction in dormant buds in SDs, without cold treatment is sufficient to induce GA20-oxidase and suppress the GA2-oxidase. Thus this data shows that FT1 induction positively regulates GA pathway, which itself has been shown earlier to open PD (Pandey et al., 2024). Additionally, we show that FT1 induction modulates the expression of several other genes that are associated with PD regulation which bolsters the link between FT1 regulation of PD. We think these new data added to the revised manuscript (as supp Fig. S9 and S10) addresses reviewers’ comment and thus provide explanation for FT1 link with PD regulation that is independent of SVL or LIM1. 2. In Fig. 3. Including ft1 knockout comparisons under both Cold and Variable Cold conditions would substantially strengthen the conclusions. This would clarify whether FT1 is essential for temperature-induced dormancy release and whether its reduced expression under Variable Cold is a primary cause of dormancy failure. Without this analysis, the proposed role of FT1 remains speculative. Response: As suggested by the reviewer, we show that ft1 mutant fails to undergo dormancy release even under constant cold conditions in contrast with the wild type (Pandey et al. 2024). However we also present data that shows that ft1 mutant does not undergo dormancy release also under variable cold. This data has been included in Fig 3g and Fig S11 and we hope this addresses the reviewers’ comments. 3. The authors present an interesting and valuable set of phenotypic data for abi1-1, (ft1, should be included) and the abi1/ft1 double mutant under various environmental treatments. However, the underlying regulatory mechanisms remain unclear. To better understand the genetic interactions and clarify whether ABI1 and FT1 act in the same or parallel pathways, it would be helpful to analyze the expression levels of key genes such as SVL, LIM1, FT1, and GA20ox in these mutant backgrounds. Specifically, comparing gene expressions across 11WSD, constant cold, and variable cold conditions could provide more direct evidence of the regulatory roles and relationships among these genes. This would significantly strengthen the mechanistic interpretation of the observed phenotypes under variable cold. Response: We thank the reviewer for the suggestion. The relationships between various factors e. g SVL, LIM1, FT1 and GA and ABA pathways are already published or included in the current manuscript. Our data in the manuscript shows that warm spikes do not act via SVL and LIM1 as these are regulated more or less the same in variable cold as constant cold (Fig. S5). Moreover in ft1 mutant, SVL or LIM1 expression is not altered (see below) which is in agreement with these acting upstream of FT1 and that FT1 does not regulate their expression. Also we have already shown that ABA and GA act antagonistically in dormancy regulation earlier and that ABA suppresses GA pathway in dormancy (Singh et al 2018). Thus we feel that the expression analysis would not be helpful in addressing whether abi1-1 and FT1 act in same pathway. However, for addressing whether ABI1 and FT1 act in same or parallel pathway, we refer to our genetic analysis (which we think addresses reviewer’s comment) in the manuscript. Our data shows that loss of ft1 in abi1-1 mutant background, does not alter the phenotype of abi1-1 (which is insensitivity to warm spikes). This is consistent with ABA being required for PD closure and of FT1 being subsequently required for PD opening in the response to cold. The analysis of ft1 mutant which cannot open PD in response to cold and do not undergo dormancy release further supports the key role of FT1 in PD opening. Thus when induction of FT1 is suppressed by warm spikes, closed PD cannot be opened, with resultant failure of the wild type to undergo dormancy release. Putting together these observations our data shows that ABA and FT1 converge, acting antagonistically on PD regulation (ABA closing and FT1 opening PD) as summarized below. Consequently, when ABA pathway is suppressed (leading to open PD), FT1 is no longer required and thus abi1/ft1 mutant phenocopies abi1 and this is further explained schematically in figure below. We hope that this addresses reviewers’ concern. However if the reviewer still wishes us to perform expression analysis we are happy to do that but this will not be possible within the time constraints of the revision process. Fig. 1b, Fig. S7- Could the authors clarify why the WT plant in Fig. 1b (11WSD) appears to retain its leaves, while the WT plant shown in Fig. S7 (10WSD) seems to have entered dormancy and shed its leaves? This apparent inconsistency raises some confusion - was there a mistake in photo selection or labeling? Or is there another explanation? Response: Thanks for pointing this out. Apologies for confusion. The correct picture has been added. Reviewer #2 (Remarks to the Author): Pandey et al. address how poplar buds are released from dormancy by environmental signals, thus adding to an existing body of work on this highly important physiological response of plants and the end of the winter season. The authors describe and investigate a previously insufficiently characterised mechanism, whereby poplar plants suppress bud break in response to fluctuating outside temperature. Understanding plant responses to such \"real life\" scenarios and variable environments is highly relevant, not only for our fundamental understanding of plant development, but also to explore and possibly predict plant responses to changing current and future climate scenarios. The authors investigate the expression and functional role of key regulators of bud break in poplar, including transcription factors, hormones, and mobile factors that are known as floral inducers, such as FT1. The manuscript reports on a key role for FT1 in triggering dormancy release, mediated by opening of plasmodesmata by reducing callose levels, and how short warm periods that interrupt cold phases can basically erase the effect of extended cold, resulting in suppression of bud break. The authors propose that FT1 is a key factor in this response, and that the underlying mechanism supports bet hedging, i.e. a differential response of buds to the same environmental input in order to increase reproductive success. The experiments are well presented and described. I very much enjoyed the high quality of the analysis of callose deposition at PDs, based on antibody detection using fluorescence labelling for entire meristems, and detailed surveys via electron microscopy with gold labelled antibodies! Overall, a well written manuscript which addresses dormancy and the role of plasmodesmata in bud break from a new angle, which was a pleasure to read. Nevertheless, there are a few points that the authors should consider and address to further strengthen the manuscript, which I have detailed below. 1) A key point the authors make in the manuscript is that FT1 expression is sufficient and can subsitute for cold in inducing PD opening and dormancy release. This statement is based, among other experiments, on an inducible FT1 transgenic line, which can overcome environmental regulation and induce bud break and PD opening (judged by reduction in callose). However, the authors have analysed changes in callose accumulation at PDs several weeks after FT1 induction, so FT1 might be a rather indirect regulator of PD callose. This problem (correlation, causality) could be resolved by assaying PD callose levels shortly after FT1 induction, and before bud break. In that context, I would like to quote a sentence from the manuscript, which highlights this more: \"Altogether, these results indicate that warm spikes act via FT1 to suppress downregulation of callose which then prevents dormancy release\". It would be equally possible to argue that warm spikes act via FT1 (and possible LIM1) to prevent dormancy release, which would normally result on PD opening and callose reduction. Response: Thank you for raising this point. When we initiated the experiment we first performed a test analysis of FT1 induction at various time points. Our data (see below) indicated that after 1 week estradiol treatment FT1 induction was negligible and significant induction of FT1 was only observed around 2 weeks. Based on this result, we chose the relevant timepoint when we could observe FT1 induction i.e. 2 weeks, and also performed analysis of callose at this time point. Thus there is good temporal correlation and no real delay of several weeks between timing of significant FT1 induction and corresponding downregulation of callose level. We are happy to include this data to clarify in revised manuscript. The link between FT1 and callose is also supported by the ft1 mutant analysis which shows that in absence of FT1, callose levels fail to be reduced after cold treatment. Moreover our grafting assay (that are indicative of PD being closed or open) show that ft1 mutant buds fail to reactivate when grafted on FT1 overexpressors (added as supp Fig S6). This result also supports that ft1 mutant presumably fails to open PD and thereby also bolsters the link between FT1 mediated callose downregulation and dormancy release. Further supporting the link between FT1, callose and dormancy is our data which shows that FT1 regulates GA pathway, inducing GA20-ox and simultaneously suppresses GA2-ox both of which are connected with callose regulation. Last, FT1 regulates the expression of genes associated with PD regulation (see below). We hope our clarification and additional data have convinced the reviewer of the causal link between FT1 with PD regulation, and our conclusion on how warm spikes act via suppression of FT1 expression to prevent PD opening and dormancy release. We have now added new data in the revised manuscript as supp Fig. S9 and S10. 2) To determine whether defects in callose deposition/degradation could affect bud dormancy break the authors used the abi1 mutant, which was previously reported to break dormancy even in cold condition, because plasmodesmata are always open in this mutant background. They showed here that the mutant displayed dormancy break also in the variable cold condition, i.e. with warm spikes interrupting the cold period.; also, they are insensitive to the warm spikes, that inhibit dormancy break in wt condition. Determining the level of callose at PDs in the three conditions (11WSD, constant cold and variable cold) in the abi1 background is important to evaluate whether a decrease in callose accumulation at the plasmodesmata is necessary and sufficient to trigger break dormancy. Response: We thank the reviewer for their comment. We have now performed analysis of callose levels in abi1-1 mutant buds at 11 weeks SDs. This new data (which is now included in revised manuscript Fig S4) (see below) shows that, already at 11 weeks under short-day conditions (11WSD), even before any cold treatment, callose levels in the abi1-1 mutant are significantly lower than in wild-type plants. This is also consistent with our previous findings which show that PD are already open in abi1-1 (Tylewicz et al., 2018). Also we have shown earlier, abi1-1 buds grafted FT1 overexpressors are able to undergo bud break unlike wild type buds which further show that PD are open in abi1-1 mutant. Our data shows that warm spikes act by preventing PD opening by callose downregulation. However new data on callose analysis of abi1-1 as well as previously published data (Tylewicz et al., 2018) show that callose levels are already low in abi1-1 mutant and PD are open and thus regulation of PD opening is uncoupled from cold regulation in abi1-1 mutant and this correlates with the insensitivity of abi1-1 to warm spikes and its phenotype. The new and previous data on abi1-1 thus strongly suggests that since PD are already open, variable cold which basically acts by suppressing the opening of closed PD is not effective in abi1-1 (as suppression of ABA response will prevent PD closure). We hope that this addresses reviewer’s comment. However, if absolutely necessary, we can check callose levels in abi1-1 at PD under cold and variable cold conditions. 3) One feature to assess bud dormancy is the presence of lipid droplets. As a matter of fact, the authors nicely observed lipid droplets in the 11WSD treatment. The information whether also buds from variable cold condition showed lipid droplets is missing (lipid droplets of what??) Response: As suggested by the reviewer, we did perform the analysis of lipid droplets (see below, Fig 5). In our hands, the lipid droplets were present not only in 11WSD but also in cold as well as variable cold throughout the bud i.e. in dormant as well as non-dormant buds. Since we find lipid droplets in 11 WSD, cold as well as variable cold, their analysis is not informative and we would therefore prefer not include them in the manuscript. 4) A reported pathway to trigger dormancy release relies upon the simultaneous downregulation and upregulation of SVL and LIM1, respectively. The authors therefore assayed expression of SVL and LIM1 in entire buds by qRT-PCR in the three different conditions. They point out that under both continuous cold and warm spiked conditions, SVL is still repressed, and LIM1 is still induced. FigS4 shows, in my opinion, a strong difference in the response of LIM1 under the two key conditions. Is that statistically different? Do all cells in a bud behave in the same manner, i.e., could there be regional differences in expression levels? A possible interpretation of the data, which differs from the authors point of view, could be that LIM1 is not sufficiently induced in the warm-spiked situation, and therefore dormancy is not broken. Response: To clarify, LIM1 expression may appear to be reduced under warm spikes, but following reviewer’s suggestion we performed statistical analysis and this reduction is statistically not significant. Therefore we feel that it is unlikely that the reduction of LIM1 contributes to warm spike effects on bud dormancy. As for regional differences, when bud dormancy is released and growth initiated, to our knowledge there is no regional difference in growth response as whole bud swells and hence we feel that investigating regional differences in LIM1 expression are likely to be non-informative and somewhat out of scope of this manuscript. 5) FT1 has been previously reported to be a cold induced promoter of dormancy release. To determine whether inhibition of dormancy release in variable cold condition was due to a failure in triggering FT1 expression, the authors performed qRT-PCR on entire buds, testing FT1 expression in the three conditions. They observed a decrease in FT1 expression in variable cold condition compared to the constant cold, suggesting that the failed dormancy release in variable cold condition was caused by reduced levels of FT1 expression in the buds. Statistical analysis would strengthen this dataset; however, given that LIM1 expression levels were also affected in variable cold conditions (compared to constant cold), focussing only on FT1 now seems to ignore other possible pathways and scenarios. Response: We agree, FT1 reduction is statistically reduced in warm spikes (indicated now) whereas LIM1 is not (see above). This is clearly indicated in the revised manuscript. 6) The authors showed here that the ft1 mutant buds do not show a decrease in callose (associated to the number of gold particles) in cold condition. Different callose levels could also be caused by a different number of PDs. Analyzing the number of plasmodesmata in ft1 mutant would be important to determine how FT1 act in dormancy release. Furthermore, the data presented here are in contrast to data presented by André et al., 2022, which uncouple FT1 function and callose presence at PDs in poplar (DOI: 10.1016/j.cub.2022.05.023). This should be discussed. Response: We agree that the number of PD is important, especially when buds are not in a dormant state, as it can affect the trafficking of information between cells. We have now included additional data showing PD density in the ft1 mutant under both 11WSD and 4WC conditions. Our analysis shows that there is no significant difference in PD density between the ft1 mutant and wild-type plants under these conditions. This data is included in revised manuscript (Fig. S7) and discussed. Thanks for pointing out the Andre et al. paper. To clarify, in contrast with what we have done here, Andre et al did not measure PD associated callose levels in ft1 mutant. However their as well as (our unpublished) grafting data clearly indicates that PD are not open i. e. ft1 buds grafted on FT1 overexpressors fail to induce bud break in line with PD being closed when FT1 is inactivated. Thus clearly in ft1 mutant PD do not open which would be in agreement with a failure to downregulate callose levels and we have now indicated this in results section as suggested. 7) The authors showed that the double mutant abi1-1/ft1 presented bud break even in variable cold condition. They concluded that warm spikes act via FT1 to suppress downregulation of callose to prevent bud break. I think that their conclusions are not supported by the data and additional observations are needed. Murata et al., (2025) very recently reported that cold triggers ABA signaling that in turn induces callose synthesis by upregulation of CALLOSE SYNTHASE genes, restricting FT translocation to the SAM in Arabidopsis thaliana. The authors here suggested a direct activity of FT1 on degradation/deposition of callose, modulating dormancy break. Analysis of FT1 translocation in abi1-1 mutant would be important to address whether movement of FT1 in the bud is important for dormancy break or FT1 solely act in modulating callose distribution at PDs. Do expression patterns of FT1, and local decrease of callose overlap, or does this occur in different, separate cells? I.e. is FT1 transport required for the observed effects? Response: Thank you for pointing out the very nice Murata et al paper. We have referred to their work and compared it with ours in revised discussion. The findings of Murata et al are completely in line with our previously published data showing that ABA is required for callose deposition whereas blocking ABA response suppresses callose accumulation and keeps PD open (Tylewicz et al 2018) as well as new data on callose analysis in abi1-1 buds (Fig. S4). However where trees differ from Arabidopsis is that in tree buds, short days induce ABA and this induces callose deposition to close PD whereas in Arabidopsis it is cold inducting ABA to close PD. Here we are looking into the process of opening of closed PD during dormancy release, a process that is mediated by cold in tree buds and all our data (genetic and callose analysis of ft1 mutant, analysis of FT1 inducible line) suggests that FT1 is induced by cold and FT1 function is essential for opening of PD and since opening of PD is essential for dormancy release, FT1 links PD opening and dormancy release. Overall, our data suggesting FT1 downregulation by warm spikes suppresses FT1 expression and this downregulation of FT1 results in suppression of callose downregulation and failure to release dormancy is supported by: ft1 mutant has high callose levels and unlike wild type, ft1 mutant fails to reduce callose levels in response to cold, conversely, FT1 induction can downregulate callose levels even without cold. Moreover new data included in revised manuscript shows that FT1 induction can regulate expression of GA pathway (that regulates callose levels) as well as that of several PD related genes. As for translocation of FT1 in abi1-1, we have already shown earlier (Tylewicz et al ., 2018) that buds of abi1-1 mutant when grafted on FT1 overexpressors are able to initiate growth, whereas wild type buds or (ft1 mutant buds) with closed PD are unable to initiate growth when grafted on FT1 overexpressors which strongly indicates the importance of FT1 mobility. As for spatial correlation between FT1 and callose, this is difficult to address as FT1 is already known to move as shown by us as well as Murata et al and thus could be both (in same as well as FT1 non-expressing cells). 8) Based on their results, the authors concluded that FT1 (together with GA) trigger dormancy break but their relationship is not further investigated. Enhanced expression of FT1 could be the effect and a consequence of callose degradation/reduced callose deposition at PDs and not the trigger of the response. Analysis of FT1 expression in the GA overexpression lines and analysis of dormancy release in GA20oxoOE ft1 doble mutant would clarify this aspect. Response: Our expression data (see above showing GA20-oxidase and GA2-oxidase expression upon FT1 induction, now also added to the revised manuscript) shows that FT1 upregulates GA20-oxidase and suppresses GA2-oxidase. Thus our data shows FT1 is upstream of GA addressing the reviewer’s comment on the relationship between FT1 and GA pathway. As for enhanced expression of FT1 being a consequence of callose reduction is not supported by our results. For example, callose levels fail to be downregulated in ft1 mutant whereas FT1 induction is sufficient to induce callose downregulation and moreover, FT1 induces GA pathway, and also impacts the expression of several PD related genes as shown above (and included in the revised manuscript). Thus these data do not favor the scenario in which callose downregulation/PD opening induces FT1 expression. Instead ,our data, together with those of others, support the scenario where FT1 downregulates callose rather than FT1 induction being a consequence of callose downregulation. 9) The authors here reported that level of expression of FT1 are lower in variable cold condition compared to cold condition; considering that the ft1 mutant presented defects in dormancy break even in cold condition they concluded that warm spikes inhibit bud break. FT1 expression was quantified by qRT-PCR on the entire bud. Analysis of the pattern of expression of FT1 would determine whether the accumulation of callose is inhibited in the same cells where FT1 expression is triggered or if FT1 acts non-cell autonomously to determine dormancy break. Response: We have tried to investigate FT1 expression in vivo by expressing FT1 its under native promoter and tagged with fluorescent protein. Despite several attempts we have been unable to express FT1 from its native promoter due to gene silencing of all the constructs from native promoter and therefore to visualize it in native conditions. However, we have shown earlier that FT1 movement is feasible and this requires open PD e. g. wild type dormant buds fail to reactivate when grafted on FT1 overexpressors whereas abi1-1 buds with open PD grafted on FT1 overexpressors can reactivate under the same conditions. Moreover, in-situ analysis by Andre et al shows that FT1 transcript is not expressed throughout the bud whereas our data shows that callose reduction is significant extensively throughout the bud. These results would suggest FT1 acts both in cells it is expressed but then FT1 movement also further amplifies callose reduction throughout the bud. Furthermore, FT1 induces GA pathway and GA itself moves via PD. Thus we feel that while knowing whether FT1 acts cell autonomously or non-cell autonomously is interesting, it is nevertheless not crucial to the key finding of the paper which is that buds decide to integrate cold by using warm spikes and that this is mediated by transcriptional regulation of FT1. 10) LIM1 regulation is one of the key responses to cold, and upregulation is reduced in variable cold conditions (Fig S4). could you provide some statistical analysis of these data? Are these qRT-PCR data? How would LIM1 mutants respond to variable cold, i.e., is LIM1 here the key factor that mediates the difference in response? Response: As suggested by the reviewer, we have performed statistical analysis and as indicated earlier, the downregulation of LIM1 by warm spikes is not statistically significant. This now indicated in the revised manuscript. This would suggest that warm spikes do not act via LIM1 downregulation supporting our data. 11) The suggested bet-hedging strategy is certainly intriguing, but not really explored sufficiently in this manuscript. As stated above (point 9), I am missing the spatial component in the analysis of bud break. Are the breaking/non-breaking buds evenly distributed at the plant? How is the response of a bud, consisting of many hundred of cells, overall integrated? I am tempted to suggest to take this part of the story out of the manuscript, and explore this in-depth in a separate manuscript. It appears here like a very interesting, but insufficiently supported add-on. Response: We thank the reviewer for positive response to our findings on variability. To clarify, in trees, lateral buds and apical buds behave differently in response to cold. Therefore, we have specifically considered analysis of apical buds for our experiments (also all other analysis was performed on apical buds). Which would mean single bud at the apex for every plant (n=20-25). Thus breaking/nonbreaking buds which would include analysis of both apical and lateral buds is not attempted in the context of this work. While we agree spatial analysis would be very useful, our findings are nevertheless highly interesting as indicated by the reviewer, to the tree community as they provide an insight into survival mechanism which has not been investigated so far. So we hope reviewer will allow us to keep this result. We are happy to incorporate the spatial aspect as suggested by the reviewer into the discussion (if necessary) as being important for the future and follow up to this work. Reviewer #5 (Remarks to the Author) This study provides important mechanistic insight into how plants integrate variable temperature signals to regulate bud dormancy release. The authors demonstrate that plasmodesmata-mediated cell–cell communication acts as a key integrator of fluctuating thermal cues, particularly through the detection and interpretation of intermittent warm spikes. They show that long warm periods suppress dormancy release by preventing PD reopening via repression of FT1 and GA20-oxidase expression. In contrast, shorter warm interruptions allow cold integration and promote dormancy release. These findings reveal a previously uncharacterized role for FT1 in callose downregulation and PD opening. The study also links temperature-responsive PD dynamics to phenotypic variability in bud break, suggesting a cellular basis for bet-hedging under unpredictable spring conditions. Overall, this work presents a compelling and original framework for understanding how plants maintain robust developmental timing amid environmental noise, with broader relevance to plant adaptation under climate change. The manuscript is well written, and the data presented are convincing, novel, and well suited for publication in Nature Communications. Below are a few comments and questions for the authors' consideration: Response: We thank the reviewer for useful suggestions to improve our manuscript. Comments: Figure 1a and Figure 4a The schematic suggests that warm spikes were applied at different times on consecutive days (e.g., from ZT20–ZT24 on the first day, and from ZT40–ZT44 on the second). Is this interpretation correct? If so, could the authors clarify the rationale for this design? Response: This is misunderstanding. In both cases, the warm spikes were applied at the same time and is indicated in materials and methods. It only appears different due to schematic figure. Figure 2, Figures S1 and S3 and discussion the observation of stronger callose deposition at PDs in the SAM compared to adjacent tissues raises an interesting possibility of cold-induced, cell–cell communication regulation. Do FT1 or GA biosynthesis genes exhibit SAM-specific expression patterns under these conditions? Could this reflect SAM-specific expression of β-1,3-glucanases involved in PD opening? Response: Perhaps there is a misunderstanding as we did not show data comparing callose levels in SAM with adjacent tissues. Nevertheless cold induced PD opening definitely favors the reactivation of cell-cell communication within the buds as is implied by our data and those of others. While we do not have exact cell resolution data, FT1 and GA biosynthesis genes are indeed expressed within the SAM (central bud) and this is true for β-1,3-glucanases. Line 119 “In agreement with prior studies, callose levels decreased in dormant buds exposed constant cold (Fig. 2a, Fig. S1a, Fig. S2a).”Could the authors clarify how the measured callose levels were specifically linked to PD openness? This connection is not immediately evident from the provided images. Response: At the tissue level, the quantification of callose levels shown in Fig. 2a and Fig. S1a (based on mean fluorescence intensity) and Fig. S2a (based on the number of gold particles per PD) reflects the well-established dynamic of high callose accumulation during dormancy and its reduction upon dormancy release. After cold treatment, callose at PD decreased to levels normally seen in actively growing shoot apices, where PD are open. This decrease coincided with dormancy release and bud break. In contrast, callose remained high in buds exposed to fluctuating cold with warm spikes, and no bud break occurred. Reduced callose therefore serves as a proxy for increased PD openness, consistent with dormancy release. Line 238 The data show that constitutive overexpression of GA20ox leads to bud break in nearly 50% of plants under variable cold conditions (Fig. S6). However, this phenotype does not fully phenocopy the effects of FT1 induction. Could the authors elaborate on potential reasons for this divergence? Does this suggest that FT1 may regulate additional GA-independent pathways required for PD opening and dormancy release? Response: Thanks for pointing this out. Indeed this result suggests that FT1 could additionally regulate a pathway that contributes to dormancy release. We have now stated this in revised discussion. Line 292-297 if cold integration mechanism that enables robust sensing of autumn to late autumn/winter transition, as, typically, there is a shift towards progressively shorter periods of warm spikes relative to cold. A lack of response to cold when the warm spikes last longer can therefore ensure that cold registration is not initiated and chilling threshold is not achieved prematurely, preventing precocious dormancy release….. (Fig.5) Response: We are not sure if there is a comment to be addressed? Is callose deposition at PDs, and the resulting closure of cell–cell communication, only triggered when warm spikes become short enough? If so, at what point in this model does PD dynamics shift from closure to opening?\" Response: To clarify, PD are closed before buds are exposed to cold. Based on the experimental conditions using 2 different fluctuating conditions, our results would suggest that the switch from close to open is initiated when warm spikes are reduced from 4 to 2 hours i. e. between 4 and 2 hours. We have now also included new data as supp Fig. S13 (see below Fig. 1) that supports this by showing that in contrast with 4 hour warm spikes when there is no significant reduction of callose, callose is significantly reduced when warm spikes are reduced to 2 hours, the latter also triggering dormancy release."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I checked the revised version and found that the authors provided a lot of new data and revised the manuscript properly, which addressed all my concerns. Regarding the title, I understand the author's explanation and agree to retain the original title for this revised manuscript.", "reviewer_2": "Thank you for the new version of the manuscript. I feel that the authors have addressed all concerns that were raised by me and the other reviewers.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "Dear Authors, The revised version shows significant improvement, including clearer explanations and the addition of new supporting data that strengthen the overall work. In my opinion, the manuscript now meets the journal’s standards and should be accepted for publication."}, "author_response": ""}]

Decision letter