Phytochrome B integrates jasmonic acid and warm temperature signaling pathways to regulate cotyledon chloroplast development

Domain: chemistry
Published: 2026-03-09
Editorial outcome: major_revision

Abstract
Chloroplast biogenesis and development are coordinated by both endogenous factors and environmental cues. The interplay between jasmonic acid (JA) and temperature in regulating chloroplast development remains poorly understood. Here, we demonstrate that methyl jasmonate (MeJA) and warm temperature (28℃) treatments additively impair chlorophyll accumulation and chloroplast development in Arabidopsis thaliana cotyledons. We found that the thermosensor phytochrome B (phyB) suppresses, whereas the JA receptor CORONATINE INSENSITIVE 1 (COI1) promotes, MeJA- and warm temperature-mediated chloroplast development. Moreover, phyB directly interacts with JASMONATE ZIM-DOMAIN 1 (JAZ1) and JAZ3, preventing their JA-induced degradation, and warm temperature attenuates this interaction. Strikingly, we reveal that transcription factors ELONGATED HYPOCOTYL 5 (HY5) and MYC2 oppositely regulate cotyledon chloroplast development in response to MeJA and warm temperature by directly and differentially modulating downstream transcriptional networks. Our study establishes a molecular framework in which phyB integrates JA and warm temperature signaling through the HY5-MYCs transcriptional regulatory network to fine-tune chloroplast development, highlighting a plant strategy for ecological adaptation.

Introduction
Global climate change poses a threat to world agriculture (Lobell et al., 2011). Modeling suggests that every 1 ℃ increase in global temperature could reduce the yield of major crops by 3-8% (Zhao et al., 2017). Plants have evolved sophisticated sensory and signaling networks to perceive and integrate environmental stimuli, such as light and temperature, to optimize growth and development. Non-stressful warm temperatures profoundly reshape plant development, accelerating phenological transitions like flowering and leaf senescence, and promoting hypocotyl and petiole elongation, a process termed thermomorphogenesis (Quint et al., 2016; Casal and Balasubramanian, 2019). Phytochrome B (phyB) functions as both a dominant red-light photoreceptor and a thermosensor that triggers light and warm temperature signaling pathways (Li et al., 2011; Casal and Balasubramanian, 2019). PhyB exists in two photoconvertible conformations: the red light-absorbing inactive Pr form and the far-red light-absorbing active Pfr form. Upon light activation, the Pfr form of phyB translocates to the nucleus, where it interacts with PHYTOCHROME-INTERACTING FACTORs (PIFs) to regulate downstream gene expression (Leivar and Quail, 2011). phyB also undergoes light-independent Pfr-to-Pr dynamic thermal reversion and the relative amount of the Pr form is accelerated with increasing temperature (Klose et al., 2020). A recent study revealed that while light induces phyB photobody formation, the N-terminal extension of phyB acts as a biophysical modulator of phase separation to directly sense warm temperature (Chen et al., 2022). This temperature-dependent thermal reversion provides the molecular basis for phyB to function as a thermosensor controlling plant elongation growth (Jung et al., 2016; Legris et al., 2016). PIF4 serves as a transcriptional hub in the warm temperature signaling pathway, with warm temperature enhancing both PIF4 transcription and PIF4 protein stability (Koini et al., 2009; Foreman et al., 2011; Kim et al., 2020). Some studies also indicate that an effect of elevated temperature on cotyledon and leaf morphology. For example, PIF4 and PIF5 are required for warm temperature-induced leaf senescence, as their double mutant retains more chlorophyll than wild-type seedlings (Kim et al., 2020).

Jasmonic acid (JA) is a phytohormone that regulates plant growth and defense against abiotic stresses (Hong et al., 2024). The canonical JA signaling pathway comprises the receptor (CORONATINE INSENSITIVE 1, COI1), repressors of the JASMONATE ZIM-DOMAIN (JAZ) family, and downstream transcription factors/regulators (Li et al., 2022a). Under normal conditions, endogenous bioactive JAs are maintained at a low basal level. Upon environmental or developmental stimulation, the F-box protein COI1 recruits JAZ proteins for ubiquitination and degradation via the 26S proteasome pathway, thereby releasing downstream transcription factors to activate JA responses (Xu et al., 2002; Chini et al., 2007; Fernandez-Calvo et al., 2011; Wang et al., 2021; Hu et al., 2023). JA signaling also inhibits growth and promotes senescence, reflecting the classic trade-off between growth and defense (Li et al., 2022a). Previous studies indicate that the basic helix-loop-helix (bHLH) transcription factors MYC2, MYC3, and MYC4 may mediate JA-induced chlorophyll degradation by directly activating chlorophyll catabolic genes (Zhu et al., 2015). Following JA treatment, the myc2/3/4 triple mutant exhibits a stay-green phenotype with high chlorophyll content, similar to the coi1-1 mutant. In contrast, lines overexpressing MYCs display precocious leaf senescence, supporting a role for JA in promoting chloroplast maturation (Qi et al., 2015; Zhu et al., 2015). Additionally, JA activates a subset of senescence-associated genes (SAGs) to promote leaf senescence. MYC2/MYC3/MYC4 activate the expression of SAG29 to enhance JA-induced leaf senescence, whereas several other bHLH transcription factors bind to the SAG29 promoter and repress its MYCs-activated expression (Qi et al., 2015).

Several previous studies have revealed connections between warm temperature and JA signaling. Warm temperature can upregulate the expression of Jasmonate Oxidases and Sulfotransferase Family 2A (ST2A) to regulate jasmonate catabolism (Zhu et al., 2021). The resulting reduction in bioactive JA levels stabilizes JAZ proteins, thereby ultimately promoting plant growth in warm temperature (Zhu et al., 2021). Similarly, neighbor competition promotes plant growth by upregulating ST2A to reduce bioactive JA levels, a process mediated through the phyB-PIF module (Fernandez-Milmanda et al., 2020).

While the majority of thermomorphogenesis studies have focused on hypocotyl and stem elongation, this study investigates cotyledon and chloroplast development. We found that warm temperature and JA additively induce cotyledon chlorosis. We demonstrate that phyB interacts with JAZ1 and JAZ3 and enhances their protein stability. Furthermore, we show that HY5 and MYC2 oppositely regulate downstream gene expression and, subsequently, JA- and warm temperature-mediated chloroplast development in the cotyledons. Our findings reveal a previously uncharacterized crosstalk between JA and warm temperature signaling pathways in regulating seedling development.

Results
JA and warm temperature additively regulate chlorophyll accumulation and chloroplast development in cotyledons

To establish a cotyledon-based experimental system, Columbia-0 (Col-0) wild-type seedlings of Arabidopsis thaliana were grown on Murashige and Skoog medium (MS) supplemented with or without methyl jasmonate (MeJA, a JA derivative) under continuous white light at temperatures ranging from 18℃ to 30℃ for 6 days. Both 25 μM and 50 μM of MeJA treatments significantly reduced the chlorophyll content in the cotyledons, and increasing temperature enhanced the chlorosis phenotype induced by MeJA (Supplementary Fig. 1). In the subsequent experiments, we examined the physiological responses by growing seedlings on MS medium in the absence (mock) or presence of 25 μM MeJA at 22℃ (normal growth temperature) and 28℃ (warm temperature). These four conditions were designated as 22℃, 22℃+MeJA, 28℃, and 28℃+MeJA (Fig. 1a).

We found that MeJA treatment at 22℃ reduced cotyledon size, chlorophyll content, maximum quantum yield of photosystem II (Fv/Fm), effective quantum yield of photosystem II (ΦPSII), electron transport rate (ETR), and qP (coefficient of photochemical quenching). The combined 28℃+MeJA treatment further significantly enhanced these effects compared to the other three conditions (Fig. 1b-e; Supplementary Fig. 2). These changes in photosynthetic fluorescence prompted us to further investigate their impact on chloroplast development. We subsequently examined chloroplast morphology using confocal microscope and thylakoid ultrastructure via transmission electron microscopy (Fig. 1f, g). Although the number of chloroplasts per cell did not differ substantially among the four conditions (Fig. 1h), chloroplast size, chlorophyll fluorescence intensity, and the number of stacked thylakoids per granum were markedly reduced under 22℃+MeJA and 28℃ compared to 22℃ alone. These parameters showed further significant reductions under 28℃+MeJA condition (Fig. 1i-k). Collectively, these results demonstrate that JA and warm temperature act additively to impair chlorophyll accumulation and chloroplast development in the cotyledons.

phyB and COI1 play antagonistic roles in JA/warm temperature-mediated cotyledon chloroplast development

Given that COI1 and phyB act as the JA receptor and a thermosensor, respectively, we investigated their roles by examining loss-of-function mutants (coi1-8 and phyB-9) and overexpressing lines (35S:COI1-GFP and 35S:Myc-phyB) under the four experimental conditions. We found that coi1-8 mutant seedlings were insensitive to MeJA and warm temperature, retaining relatively green cotyledons, higher chlorophyll levels, higher Fv/Fm, ΦPSII, and qP compared to Col-0 under the 28℃+MeJA condition, whereas 35S:COI1-GFP plants were largely similar to the wild type (Fig. 2a-d; Supplementary Fig. 3a-d). Although phyB-9 seedlings exhibited smaller cotyledons than the other genotypes (Supplementary Fig. 3a), they displayed opposite phenotype to coi1-8 at 28℃+MeJA (Fig. 2a-d; Supplementary Fig. 3b-d). In contrast, 35S:Myc-phyB seedlings showed greener cotyledons with slightly increased Fv/Fm relative to Col-0 (Fig. 2a-d). Consistent with these observations, coi1-8 and 35S:Myc-phyB lines exhibited significantly larger chloroplast area and stronger chlorophyll autofluorescence compared to Col-0, whereas phyB-9 and 35S:COI1-GFP lines displayed opposite phenotypes under the 28℃+MeJA condition (Fig. 2e-h). Additionally, coi1-8 mutants had a greater number of chloroplast per cell than Col-0 at 28℃+MeJA (Fig. 2g).

Since phyB also functions as a classical red-light photoreceptor, we further examined the seedling phenotype under red light. Similar to the results under white light, phyB-9 cotyledons showed enhanced chlorosis compared to the wild type following combined warm temperature and MeJA treatment (Supplementary Fig. 3e, f). Together, these results indicate that phyB suppresses, whereas COI1 promotes, the JA- and warm temperature-mediated inhibition of cotyledon chloroplast development.

phyB physically interacts with JAZ1 and JAZ3

JAZ proteins serve as central repressors in the JA signaling pathway (Hu et al., 2023). In our previous work, we generated green fluorescent protein (GFP)-fused transgenic lines for several JAZ genes and demonstrated that JAZ3 regulate seedling morphogenesis in response to light and warm temperature (Huai et al., 2024). Based on this, we hypothesized that phyB might functionally interact with JAZ proteins. To test this, we created homozygous 35S:JAZ1-GFP phyB-9 and 35S:JAZ3-GFP phyB-9 lines through genetic crossing. Under the 28℃+MeJA condition, both 35S:JAZ1-GFP phyB-9 and 35S:JAZ3-GFP phyB-9 seedlings largely retained green cotyledons and displayed chlorophyll contents comparable to their respective overexpression lines (Supplementary Fig. 4). This suggests that JAZ1 and JAZ3 act largely downstream of phyB in regulating cotyledon chlorophyll accumulation.

We next conducted a series of experiments to investigate their potential interaction. First, a yeast two-hybrid assay showed that while full-length (FL) phyB (fused with the GAL4 activation domain, GAD) interacted only weakly with JAZ1 (fused of the GAL4 DNA-binding domain, GBD), the C-terminal fragments of phyB (C651 or C451) strongly interacted with both JAZ1-GBD and JAZ3-GBD (Fig. 3a). Second, a luciferase complementation imaging (LCI) assay in Nicotiana benthamiana leaves revealed that co-expression of either JAZ1-nLUC (N-terminal half of firefly luciferase) or JAZ3-nLUC with phyB-cLUC (C-terminal fragment of luciferase) produced strong luminescence signals compared to controls (Fig. 3b). Third, bimolecular fluorescence complementation (BiFC) assays in Arabidopsis protoplasts showed that co-transformation of phyB-nYFP (N-terminus of yellow fluorescent protein) and JAZ1-cYFP (C-terminal half of YFP) or JAZ3-YFP reconstituted YFP fluorescence specifically to the nucleus (Fig. 3c). Furthermore, we performed co-immunoprecipitation (Co-IP) experiments using 35S:JAZ1-GFP and 35S:JAZ3-GFP transgenic lines, with or without 35S:Myc-phyB. When plants were grown at 22℃, Myc-phyB was co-precipitated with anti-GFP beads in samples from 35S:JAZ1-GFP 35S:Myc-phyB and 35S:JAZ3-GFP 35S:Myc-phyB seedlings. This interaction was markedly reduced after transferring the plants to 28℃ for 3 h (Fig. 3d, e), indicating that warm temperature attenuates the phyB-JAZ association. Finally, an in vitro pull-down assay confirmed that Glutathione S-transferase fused to phyB (GST-phyB-C1), but not GST alone, could pull down His-tagged JAZ1 and His-JAZ3 (Fig. 3f, g). Together, these results demonstrate that phyB directly interacts with JAZ1 and JAZ3, and that warm temperature can weaken this interaction.

phyB regulates the protein stability of JAZ1 and JAZ3

The interaction between phyB and JAZ1/JAZ3 led us to investigate how phyB influences the properties of these JAZ proteins. Since JA promotes the ubiquitination and subsequent 26S proteasome-dependent degradation of JAZ proteins (Thines et al., 2007), we examined whether phyB affects the ubiquitination of JAZ1/JAZ3 under exogenous MeJA treatment. To capture transient ubiquitination signals, the proteasome inhibitor Bortezomib (Btz) was included to allow accumulation of poly-ubiquitinated proteins. Six-day-old 35S:JAZ1-GFP and 35S:JAZ1-GFP phyB-9 plants were grown at 22℃ or 28℃ and treated with 100 μM MeJA for an additional 3 h at the respective temperature. After MeJA treatment and immunoprecipitation with anti-GFP beads, ubiquitinated JAZ1 levels were strongly increased in 35S:JAZ1-GFP samples at both temperatures, as expected (Fig. 4a, b). Notably, ubiquitinated JAZ1 levels were further elevated in 35S:JAZ1-GFP phyB-9 plants compared to 35S:JAZ1-GFP at 22℃, but this difference was not apparent at 28℃ (Fig. 4a, b). This suggests that phyB inhibits JAZ1 ubiquitination in a temperature-dependent manner. Similar results were observed for JAZ3 ubiquitination (Supplementary Fig. 5a, b).

We next examined how phyB regulates JAZ1/JAZ3 degradation. First, the addition of MG132 (a 26S proteasome inhibitor) blocked MeJA-induced degradation of JAZ1 and JAZ3 in 35S:JAZ1-GFP and 35S:JAZ3-GFP plants, respectively, at 22℃. Moreover, JAZ1-GFP and JAZ3-GFP protein levels were lower in the phyB-9 mutant background (35S:JAZ1-GFP phyB-9 and 35S:JAZ3-GFP phyB-9, respectively) (Fig. 4c; Supplementary Fig. 5c). Second, time-course degradation experiments showed that JAZ1-GFP and JAZ3-GFP levels gradually decreased in 35S:JAZ1-GFP and 35S:JAZ3-GFP seedlings, respectively, upon MeJA treatment at 22℃. This degradation was accelerated in the corresponding phyB-9 mutant lines (Fig. 4d; Supplementary Fig. 5d). When the same treatments were applied at 28℃, the differences in MeJA-triggered degradation of JAZ1 between 35S:JAZ1-GFP and 35S:JAZ1-GFP phyB-9, and of JAZ3 between 35S:JAZ3-GFP and 35S:JAZ3-GFP phyB-9, were less pronounced than those observed at 22℃ (Fig. 4e, f; Supplementary Fig. 5e, f). Consistently, JAZ1-GFP and JAZ3-GFP protein levels were substantially reduced in the phyB-9 background under 22℃, 22℃+MeJA, and 28℃ conditions, but the reduction was less marked under 28℃+MeJA (Supplementary Fig. 6a, b). Furthermore, we purified recombinant His-JAZ1 and His-JAZ3 proteins and performed an in vitro cell-free degradation assay. A time-dependent decrease in JAZ1 and JAZ3 was observed in Col-0 extracts, and this reduction was significantly delayed in extracts from 35S:Myc-phyB plants in the presence of ATP (Supplementary Fig. 6c, d). Together, these results indicate that phyB suppresses JA-induced degradation of JAZ1/JAZ3 at normal temperature, but this suppressive effect is attenuated under warm temperature conditions.

HY5 and MYC2/MYC3/MYC4 transcription factors oppositely regulate JA/warm temperature-mediated responses

We hypothesized that certain downstream transcription factors in the warm temperature and JA signaling pathways might be involved in regulating cotyledon and chloroplast development. HY5 and PIFs are major components of the phyB pathway (Toledo-Ortiz et al., 2014; Liu et al., 2017; Xiao et al., 2022), while MYC transcription factors play central roles in JA-dependent seedling morphogenesis (Ortigosa et al., 2020; Song et al., 2022). We performed phenotypic analyses and found that no discernible morphological differences were observed between these mutants and the wild type under 22℃, 22℃+MeJA, or 28℃ alone (Supplementary Fig. 7; Supplementary Fig. 8). Strikingly, after 28℃+MeJA treatment, while the three pif mutants (pif1 pif3, pif4-1, pifq) showed mild yellowing and marginally reduced chlorophyll levels similar to Col-0, the hy5-215 mutant displayed severe cotyledon chlorosis with a decrease in chlorophyll content (Fig. 5a, b; Supplementary Fig. 7). Conversely, the myc2-2 and myc2/3/4 mutants retained higher chlorophyll contents than Col-0, while the 35S:MYC2-Myc seedlings exhibited a wild-type-like phenotype (Fig. 5a, b; Supplementary Fig. 8). These results suggest that HY5 and MYC2/MYC3/MYC4 regulate cotyledon development in response to combined JA and warm temperature.

We next performed detailed characterization of the hy5-215 and myc2/3/4 mutants. Under the 28℃+MeJA condition, myc2/3/4 mutants maintained significantly higher PSII photochemical efficiency, whereas hy5-215 mutants showed lower Fv/Fm compared to Col-0 (Fig. 5c, d). Consistent with this, myc2/3/4 mutants preserved intact thylakoid architecture with well-stacked grana and continuous stromal lamellae, and exhibited a greater number of chloroplasts, larger chloroplast area, and stronger chlorophyll autofluorescence than Col-0 (Fig. 5e-i; Supplementary Fig. 9a). In contrast, hy5-215 displayed hallmark features of chloroplast degeneration (Fig. 5e-i; Supplementary Fig. 9a). Furthermore, quantification of chloroplast distribution and ultrastructural integrity across mesophyll cells indicated chloroplast defects in approximately 26% of Col-0 cells, 12% of myc2/3/4 cells, and 81% of hy5-215 cells (Supplementary Fig. 9b, c).

Immunoblot analyses were performed to examine chloroplast protein homeostasis upon 28℃+MeJA treatment. Levels of Rubisco large and small subunits (RbcL/RbcS), light-harvesting complex II chlorophyll a/b-binding protein 3 (Lhcb3), and D1 (the core subunit of photosystem II) were elevated in myc2/3/4 but decreased in hy5-215 compared with Col-0. In contrast, the stress-responsive chaperon HSP70 was markedly reduced in myc2/3/4 but induced in hy5-215 (Supplementary Fig. 9d). Moreover, under prolonged warm temperature and MeJA treatment, HY5 protein levels declined, whereas MYC2 accumulated markedly (Supplementary Fig. 9e). Together, these results demonstrate that HY5 and these MYCs oppositely regulate JA and warm temperature-mediated chloroplast development in the cotyledons.

Transcriptomic analysis of genes regulated by JA and warm temperature

To elucidate the molecular basis of JA- and warm temperature-mediated inhibition of chloroplast development, we harvested cotyledons from Col-0 seedlings grown under four conditions and performed RNA-seq analysis. Transcriptome analysis revealed 1,446, 1,958, and 2,576 differentially expressed genes (DEGs; fold-change ≥2, false discovery rate (FDR) < 0.05) under the 22℃+MeJA, 28℃, or 28℃+MeJA conditions, respectively, compared with the 22℃ control; 365 DEGs were common to all three treatments (Supplementary Fig. 10a, Supplementary Data 1). Gene ontology (GO) and biological process (BP) enrichment analyses indicated that 22℃+MeJA primarily regulated genes associated with environmental stress and JA signaling (Supplementary Fig. 10b). Treatment with 28℃ alone affected genes related to abiotic stress and abscisic acid (ABA) signaling (Supplementary Fig. 10c), whereas the 28℃+MeJA combination altered genes involved in the biological processes of defense response and responses to salt stress and hypoxia (Supplementary Fig. 10d). Notably, 811 genes were specifically regulated under 28℃+MeJA; among these, 469 up-regulated genes were enriched in biosynthesis of secondary metabolites and immune response, while 342 down-regulated genes were associated with cellular function, growth, and developmental regulatory processes (Supplementary Fig. 10e, f, Supplementary Data 2). Venn analysis further confirmed that 28℃+MeJA induced a greater number of DEGs than 22℃+MeJA (Supplementary Fig. 10g, Supplementary Data 3). Specifically, 22℃+MeJA regulated genes related to abiotic stress and JA response (Supplementary Fig. 10h), whereas 28℃+MeJA shifted the profile toward cellular response to hypoxia, water deprivation, ABA, and heat (Supplementary Fig. 10i). Together, these results suggest that warm temperature and JA signal activate stress responses but suppress growth processes, which underlie the cotyledon chlorosis phenotype.

Transcriptomic analysis of genes regulated by HY5 and MYC2/MYC3/MYC4

To identify genes specifically regulated by the HY5 and MYC transcription factors, we compared transcriptomic profiles of the hy5-215 and myc2/3/4 mutants with Col-0 under the 28℃+MeJA condition. Compared to Col-0, the hy5-215 mutant showed 1,691 DEGs, with a roughly equal distribution for up- and down-regulated genes (Fig. 6a, b, Supplementary Data 4). In contrast, the myc2/3/4 triple mutants displayed 1,828 DEGs, of which 1,434 were down-regulated (Fig. 6c, d, Supplementary Data 5), suggesting that these MYC transcription factors function primarily as transcriptional activators in this context. GO annotation revealed that HY5 promotes chloroplast development by up-regulating genes involved in photosynthesis light harvesting, delaying leaf yellowing and promoting cotyledon development (Rashotte et al., 2006; Cheng et al., 2008; Kim et al., 2016). Conversely, MYC2/3/4 activated genes associated with abiotic and biotic responses, JA and ABA signaling, and particularly those involved in senescence and chlorophyll degradation (Fig. 6f). Expression patterns of 6 representative genes were validated by RT-qPCR (Supplementary Fig. 11). Additionally, HY5 predominantly repressed biological processes related to abiotic stress and ABA response (Supplementary Fig. 12a), whereas MYC2/3/4 negatively regulated pathways such as “regulation of DNA-templated transcription” and “auxin signaling pathway” (Supplementary Fig. 12b).

In addition to genes uniquely regulated by HY5 or MYCs, we also identified shared targets by both transcription factors. Among these, 126 genes were down-regulated in myc2/3/4 mutants but upregulated in hy5-215 mutants, whereas 38 genes showed the opposite patterns. Furthermore, 43 genes were co-upregulated and 340 genes were co-downregulated in both mutants (Supplementary Fig.12c; Supplementary Data 6). Given the contrasting yellowing phenotypes of myc2/3/4 and hy5-215 mutants, we focused on the 126-gene cluster. These genes were predominantly associated with stress responses, with a smaller subset linked to leaf senescence and chlorophyll catabolism (Supplementary Fig. 12d, e). RT-qPCR confirmed that three representative genes, SAG21, MES16, and RAP2.6, were consistently down-regulated in myc2/3/4 but up-regulated in hy5-215 under 28℃+MeJA treatment (Supplementary Fig. 12f). Collectively, these results establish HY5 and MYCs as central hubs that integrate warm temperature and JA signaling during cotyledon development, with HY5 promoting growth- and chloroplast-related genes, and MYCs activating stress- and senescence-related pathways.

Genome-wide identification of direct targets of HY5 and MYC2

Given that HY5 and MYC2 are transcription factors, we performed DNA affinity purification sequencing (DAP-seq) to identify their genome-wide binding sites. A total of 17,633 HY5-binding peaks were identified (Supplementary Data 7), with 3,770 peaks consistently detected across two technical replicates (Supplementary Fig. 13a). Functional distribution analysis showed that 95.87% of peaks resided in gene-associated regions, of which >80% localized to promoters, while only 4.13% were in intergenic regions (Supplementary Fig. 13b). HY5 exhibited strong binding enrichment near the transcription start sites (TSS) of target genes (Fig. 7a). For MYC2, 3,566 common binding peaks were identified (Supplementary Fig. 13c, and Supplementary Data 8). Spatial distribution analysis revealed strong MYC2 binding near TSS (Fig. 7b), predominantly within promoter regions (Supplementary Fig. 13d). De novo motif analysis identified a highly enriched 10-bp conserved sequence (“HCACGTGGCA”; E-value = 1.0e-393) for HY5 (Fig. 7c). Similarly, a significantly enriched motif (Motif 9) containing a 13-bp conserved sequence "ACACGTGHYWWAW" (E-value = 1.2e-25) was identified for MYC2 (Fig. 7d). The core motif “CACGTG” corresponds to the canonical G-box recognized by both HY5 and MYC2, confirming the reliability of the data.

To identify direct targets of HY5 and MYC2, we integrated DAP-seq and RNA-seq data. Among the putative HY5 target genes from DAP-seq data, 660 genes (262 up-regulated and 398 down-regulated) were differentially expressed in the hy5-215 mutant under 28℃+MeJA treatment (Supplementary Fig. 13e, Supplementary Data 9). These genes were primarily associated with organ development, hormone-mediated signaling pathway and DNA templated transcription (Supplementary Fig. 13f). Strikingly, 8 genes directly associated with cotyledon development were identified, and heatmap analysis showed that WAVY GROWTH 2 (WAG2) and CYTOKININ RESPONSE FACTOR (CRF3) were significantly enriched among HY5 targets (Fig. 7e). In addition, 8 representative genes responsive to stress and hormone signaling were selected for visualization (Fig. 7f). Similarly, by comparing DEGs in myc2/3/4 mutants with MYC2 binding sites, we identified 202 potential direct target of MYC2 (33 up-regulated and 169 down-regulated) (Supplementary Fig. 13g, Supplementary Data 10). GO analysis revealed that many stress-responsive pathways ranked among the top ten enriched terms (Supplementary Fig. 13h), consistent with the role of MYC2 in JA-mediated cotyledon chlorosis. Heatmap analysis revealed that 9 genes involved in leaf morphogenesis and 5 JA-responsive genes were enriched among MYC2 targets (Fig. 7g). Genome-wide distribution analysis detected MYC2 occupancy peaks in the promoter regions of Related to AP2 2.6 (RAP2.6) (Supplementary Fig. 13i). Together, these analyses demonstrate that HY5 and MYC2 target a broad set of downstream genes related to cotyledon development and JA response.

HY5 and MYC2 directly activate the expression of chloroplast development-related genes

We then selected several representative downstream target genes to validate the DAP-seq findings. Golden-Like 1 (GLK1) is a well-known transcription factor that controls chloroplast development (Li et al., 2025). HY5 fused to the activation domain (AD-HY5) and drove the expression of the LacZ reporter under the control of the GLK1, GLK2, WAG2, COLD-REGULATED 15B (COR15B), 9-cis-epoxycarotenoid dioxygenase 3 (NCED3), or LOW-TEMPERATURE-INDUCED 65 (LTI65) promoter in yeast cells (Supplementary Fig. 14a). Similarly, AD-MYC2 directly bound to the promoter of NON-YELLOW COLORING 1 (NYC1), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), MYB47, Acyl-CoA-Binding Protein 3 (ACBP3), HSP101 or RAP2.6 and activated reporter expression (Supplementary Fig. 14b). Promoter analysis identified the presence of multiple motifs in the GLK1 promoter (Fig. 8a). To examine whether HY5 binds the promoter in vivo, we performed chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) using a 35S:HY5-GFP transgenic line. The result showed that HY5 directly associated with the GLK1 promoter at the 22℃ or 28℃ conditions. Notably, this binding was attenuated under 28℃+MeJA treatment (Fig. 8b). In contrast to HY5, MYC2 bound specifically to the RAP2.6 promoter only under the 28℃+MeJA condition, with no significant binding detected at 22℃ or 28℃ alone (Fig. 8c, d). These results demonstrate that HY5 preferentially binds its target genes under normal growth condition, whereas MYC2 binding occurs predominantly under combined warm temperature and JA treatment.

To assess the transcriptional activation capacity of HY5 and MYC2 on their target genes, we conducted a transient expression assay in Arabidopsis protoplasts. Effector constructs (35S:HY5-HA, 35S:MYC2-HA, or an empty vector control) were co-expressed with reporter constructs in which the LUC gene was driven by various promoters. HYS5-HA significantly activated LUC expression under the control of GLK1, WAG2, COR15B, or LTI78 promoter compared with the vector control (Fig. 8e, f). MYC2-HA strongly activated LUC expression driven by the NYC1, SAG20, RAP2.6, or SAG21 promoter (Fig. 8g, h). Further analysis revealed that HY5 transcriptional activity was reduced in the phyB mutant background, whereas MYC2 activity remained independent of COI1 (Supplementary Fig. 15a, b). We next examined whether warm temperature and JA influence the function of HY5 and MYC2. Protoplasts expressing the effector-reporter constructs were treated with or without 10 μM MeJA for 3 h at 22℃ or transferred to 28℃ for the same period before luciferase measurement. HY5-mediated activation of WAG2 was consistently observed under 28℃ or MeJA alone and was further enhanced under 28℃+MeJA (Supplementary Fig. 15c). MYC2-driven activation of RAP2.6 was markedly enhanced under 28℃ or 28℃+MeJA conditions (Supplementary Fig. 15d). Together, these findings demonstrate that HY5 and MYC2 function as key transcription factors integrating warm temperature and JA signaling during chloroplast development.

Methods
Plant materials and growth conditions

The Arabidopsis wild type is of the Columbia (Col-0) ecotype. The phyB-9 (Reed et al., 1993), hy5-215 (Oyama et al., 1997), pif1 pif3 (Li et al., 2023), pif4-1 (Huai et al., 2018), pifq (Zhang et al., 2014), coi1-8 (Xu et al., 2002b), myc2-2 (SALK_083483), my2/3/4 (Fernández-Calvo et al., 2011), 35S:COI1-GFP, 35S:JAZ1-GFP, 35S:JAZ3-GFP (Huai et al., 2024), 35S:MYC2-Myc (An et al., 2017), 35S:Myc-phyB (Liu et al., 2021), and 35S:HY5-GFP (Jing et al., 2020) lines were reported previously. All mutants were validated using PCR genotyping and sequencing. The double mutant/transgenic lines were generated by genetic crosses and verified via phenotypic assessment, antibiotic selection, PCR genotyping and/or sequencing. The seeds were surface-sterilized and sown on Murashige and Skoog (MS) medium with different concentrations of MeJA (392707, Sigma-Aldrich) containing 1% sucrose and 0.6% agar. The seeds were subsequently stored in darkness at 4℃ for 3 days, and then exposed to continuous white light (40 μmol m-2 s-1) or red light (40 μmol m-2 s-1) at either 22℃ or 28℃ for 6 days using light-emitting diodes. The adult plants were grown in proximity in soil within a growth chamber, under long-day conditions (16 hours light / 8 hours dark), with 60-70% humidity at 22 ± 2℃. Phenotypes were documented using a stereomicroscope (Nikon, http://www.nikon.com). Cotyledon area was measured for at least 20 individual seedlings using ImageJ software (http://rsbweb.nih.gov/ij/).

Chlorophyll measurement

Chlorophyll measurement was conducted following the previously described method (Zhang et al., 2017). Briefly, twenty seedlings were weighed and subsequently extracted with 80% acetone in the dark for a duration of 6 hours. The absorbance was measured at 663 nm and 647 nm, and total chlorophyll contents was calculated as (7.15×A663+18.71×A647) and expressed as mg per gram fresh weight (mg/g FW).

Chloroplast morphology analysis

For confocal laser scanning microscopy, cotyledons of 6-day-old seedlings were excised and mounted on glass slides. Microscopic imaging was performed using an Olympus FV1000MPE multiphoton fluorescence lifetime system equipped with a 63× water-immersion objective. Fluorescence detection parameters were set as excitation wavelength (488 nm) and emission spectral collection (490-585 nm). Images were acquired using a Z-stack scanning mode (0.5 μm step size) and processed via multi-focal plane image fusion. Subsequent image analysis and data export were conducted using FV10-ASW software (v4.2). For quantitative analysis, ImageJ software was employed to evaluate chloroplast density (number per cell area), mean chloroplast area, and mean chlorophyll autofluorescence intensity normalized to the corresponding chloroplast area.

Chlorophyll fluorescence imaging

To assess photosynthetic efficiency, seedlings were dark-adapted for 30-minute prior to measurements. Chlorophyll fluorescence imaging system (Imaging-PAM, WALZ) was used to determine the maximum quantum yield of photosystem II (ΦPSII) and effective quantum yield of PSII (Fv/Fm), and qP (coefficient of photochemical quenching). The ETR and redox state of the Q electron acceptor of PSII (1-qP) were analyzed as previously described (Liu et al., 2024). During the experiment, a continuous actinic light (80 μmol m⁻² s⁻¹) was applied as the background illumination. To determine the maximum fluorescence yield under light-adapted conditions (Fm'), the system delivered saturating pulse light (2800 μmol m⁻² s⁻¹) at 20-second intervals. All fluorescence parameters were acquired and processed using ImagingWin software.

Transmission electron microscopy

The samples were fixed overnight at 4℃ in a solution containing 3% (w/v) glutaraldehyde in 0.1 M Phosphate-Buffered Saline (PBS), pH 7.2. After fixation, they were washed three times (30 min each) with 0.1 M PBS buffer and post-fixed with 2% osmium tetroxide (OsO₄) in the same buffer at 4℃ for 3 h. For dehydration, the samples were treated with a graded ethanol series (50%, 70%, and 90% ethanol, 30 min each) This was followed by two changes of anhydrous ethanol (30 min each, RT). Next, the samples were transitioned into acetone (two changes, 30 min each) and infiltrated with Quetol-651 epoxy resin (Nisshin EM, Tokyo, Japan) using a 50:50 acetone: resin mixture (3 h), followed by pure resin (3 h). Polymerization was carried out at 60℃ for 24 h. Ultrathin sections (80 nm thick) were cut using an ultramicrotome (Ultracut UCT, Leica) equipped with a diamond knife and mounted onto copper grids. The sections were stained with 1% uranyl acetate (15 min, RT), rinsed with distilled water, and then counterstained with lead citrate (3 min, RT) (Sigma-Aldrich). TEM imaging was performed using a JEOL JEM-1230 microscope (80 kV acceleration voltage). Digital images (3296× 2472 pixels) were acquired with a CCD camera (EM-14830RUBY2, JEOL). Microscopy observations were conducted by Tokai Electron Microscopy (Nagoya). Ultrastructural analysis was performed on transmission electron micrographs to quantitatively assess chloroplast morphology. To ensure statistical robustness, at least 50 grana were randomly selected and analyzed per condition from a minimum of 10 chloroplasts. All micrographs were captured at a standardized magnification and calibrated using an internal scale bar. The thylakoid stacking number per granum quantified on a per-chloroplast-profile basis using ImageJ software.

Plasmid construction

The coding sequences or fragments of phyB, JAZ1, JAZ3, HY5, and MYC2 were amplified from Col-0 genomic DNA or cDNA using KOD DNA Polymerase (TOYOBO). The resulting DNAs were cloned into the pEASY-Blunt Simple vector (CB111, TransGen) and verified by sequencing. Various plasmids and vectors were digested with the appropriate restriction enzymes, ligated and transformation into E. coli Trans-T1 (TransGen) chemically competent cells.

For yeast one-hybrid assay, promoter fragments of GLK1, GLK2, WAG2, COR15B, LTI65, NCED3, NYC1, SOC1, MYB47, HSP101, ACBP3, and RAP2.6 were amplified from Col-0 genomic DNA and inserted into the EcoR1-SalI sites of pLacZi-2μ (Lin et al., 2007) using a Seamless Assembly Cloning kit (C5891-100, CloneSmarter), to generate pGLK1:LacZ, pGLK2:LacZ, pWAG2:LacZ, pCOR15B:LacZ, pLTI65:LacZ, pNCED3:LacZ, pNYC1:LacZ, pSOC1:LacZ, pMYB47:LacZ, pHSP101:LacZ, pACBP3:LacZ, and pRAP2.6:LacZ, respectively. To obtain AD-HY5, AD-MYC2, their coding sequences were amplified and cloned into the EcoRI-XhoI sites of pB42AD/pJG4-5 (hyykb101, Clontech).

For yeast two-hybrid assay, the full-length JAZ1/JAZ3 sequences were released from their respective pEASY plasmids using EcoRI and SalI, and inserted into the EcoRI-SalI sites of the pGBKT7 vector (PT3248-5, Clontech) to generate JAZ1-GBD, and JAZ3-GBD, respectively. The phyB-GAD, phyB-C4A51-GAD, and phyB-C651-GAD plasmids were produced as previously described (Liu et al., 2021).

For transient LCI assay, the full-length coding sequences of JAZ1/JAZ3 were amplified and inserted into the KpnI-SalI sites of pCAMBIA1300-nLUC, generating JAZ1-nLUC and JAZ3-nLUC. The phyB-cLUC plasmid was described previously (Liu et al., 2021).

For recombinant protein expression, the full-length coding sequences of JAZ1/JAZ3 were amplified and inserted into the EcoRI-SalI sites of the pCold-His-TF (3365, Takara) by recombination, resulting in His-JAZ1 and His-JAZ3, respectively. The GST-phyB-C1 plasmid was described previously (Liu et al., 2021).

For BiFC assay, the full-length phyB and JAZ1/JAZ3 were inserted into pSAT4A-nYFP or pSAT4A-cYFP to generate phyB-nYFP, JAZ1-cYFP, and JAZ3-cYFP respectively.

For transient expression in Arabidopsis mesophyll protoplasts and the transient luciferase reporter assay, MYC2 and HY5 were inserted into the pUC18-3HA vector to produce pUC18-MYC2-HA and pUC18-HY5-HA, respectively. The promoters of WAG2, GLK1, COR15B, LTI78, NYC1, SAG20, RAP2.6, and SAG21 were amplified and cloned into the pGreenII 0800-LUC vector to generate pWAG2:LUC, pGLK1:LUC, pCOR15B:LUC, pLTI78:LUC, pNYC1:LUC, pSAG20:LUC, pRAP2.6:LUC and pSAG21:LUC, respectively. All primers were listed in Supplementary Table 1.

RNA isolation and RT-qPCR

RNA isolation and RT-qPCR were conducted as described previously (Li et al., 2022c). Six-day-old seedlings were harvested and immediately frozen in liquid nitrogen. Total RNA was extracted using the SteadyPure Universal RNA Extraction Kit (Accurate Biotechnology), followed by treatment with RNase-free DNase I (Thermo Fisher Scientific). The first-strand cDNA was synthesized from 1 μg of total RNA using an oligo (dT) primer and reverse transcriptase (Invitrogen). Quantitative real-time PCR (qRT-PCR) was conducted using the cDNA samples and SYBR Green I Master (Takara) on a Light-Cycler 480 (Roche) in accordance with the manufacturer’s instructions. Data analysis and calculation were performed using the 2-ΔΔCT method based on three independent biological samples. Expression levels were normalized using UBQ1 as the internal control. The sequences of primers are provided in Supplementary Table 1.

Yeast one-hybrid assay

The yeast one-hybrid assay was performed as described previously (Liu et al., 2021). Various LacZ reporter plasmids were co-transformed with either AD-HY5 or AD-MYC2 into the yeast strain EGY48. The resulting transformants were grown on SD/-Ura-Trp dropout plates supplemented with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) for color development.

Yeast two-hybrid assay

The yeast two-hybrid assay was conducted as described previously (Li et al., 2022c). The GBD-fusion and GAD-fusion plasmids were co-transformed into the yeast strain Y2H Gold and subsequently selected on SD/-Leu-Trp (LW) dropout medium. Growth on SD/-Leu-Trp-His-Ade (LWHA) dropout medium indicated a positive protein-protein interaction.

LCI assay

The nLUC/cLUC-fusion constructs and empty plasmid were introduced into Agrobacterium strains GV3101 separately and subsequently resuspended in infiltration buffer [10 mM MgCl2, 10 mM MES (pH 5.6), 200 μM acetosyringone] to a final OD600 of 1.0-1.5. Different nLUC/cLUC-fusion constructs were mixed with an equal volume of conjugative p19 plasmid suspension. The bacteria mixtures were incubated at 28℃ for 2-3 hours and then introduced into the young leaves of 5-week-old N. benthamiana plants grown in a growth chamber. Leaves were collected 2 days after the infiltration and sprayed with 2 μM D-luciferin (dissolved in 0.02% Triton X-100, v/v) for the detection of luciferase signals using a NightSHADE LB985 plant imaging system fitted with a CCD camera (Berthold Technologies). Experiments were performed at least three times with consistent results.

BiFC assay

The BiFC assays were performed as described (Li et al., 2022c). Plasmids expressing N- and C-terminal YFP fusion proteins were co-transfected into Arabidopsis protoplasts. The protoplasts were incubated in darkness for 12 to 16 hours. Yellow fluorescent protein (YFP) signals were observed using a confocal microscope (Zeiss LSM 980).

Pull-down assay

Recombinant His-JAZ1, His-JAZ3 and GST-phyB-C1 fusion proteins were expressed in Escherichia coli BL21 (DE3) strain and induced with isopropyl β-D-thiogalactopyranoside at 16℃. 5 μg of bait protein (GST-phyB-C451 or GST) and 5 μg of prey protein (His-JAZ1/His-JAZ3) were combined with 1 mL of binding buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% (w/v) glycerol and 0.5% (v/v) Triton X-100]. After mixing, 10 μL of Glutathione Sepharose 4B beads (BE6948, EASYBIO) were added and the mixture was incubated at 4℃ for 1 hour. After washing the beads four times with washing buffer [50 mM Tris-HCl pH 7.5, 200 mM NaCl, and 1% (v/v) TritonX-100], the proteins were subsequently eluted using 2× SDS loading buffer at 95℃ for 8 min. The input and immunoprecipitation samples were separated by 8% SDS-PAGE gel, and proteins were detected by immunoblotting with anti-GST and anti-His antibodies.

Protoplasts transformation and transient luciferase reporter assay

Arabidopsis mesophyll protoplasts were isolated from 4-week-old Col-0 plants as described previously (Li et al., 2022c). Protoplasts were co-transformed with 5 μg of reporter plasmids and 5 μg of effector plasmids. After incubation in darkness for 16 hours, protoplasts were lysed, and the activities of LUC and REN were quantified using a Dual-Luciferase Reporter Assay System (E1910, Promega).

Protein immunoblotting

For immunoblotting of JAZ1/JAZ3-GFP and photosystem proteins, samples were homogenized in extraction buffer [50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 150 mM NaCl, 1 mM EDTA, 10% (w/v) glycerol, 1% (v/v) Triton X-100, 10 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonylfluoride (PMSF), 1× EDTA-free protease inhibitor cocktail (4693159001, Roche), 25 μM MG132 (474790, Sigma-Aldrich)]. For immunoblotting of HY5 and MYC2 proteins, total proteins were extracted with buffer [50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 150 mM NaCl, 1 mM EDTA, 10% (w/v) glycerol, 0.1% (w/v) Tween 20, 20 mM DTT, 1 mM PMSF, 1× EDTA-free protease inhibitor cocktail, 50 μM MG132]. Protein concentration was determined with Bradford assay reagent (Bio-Rad). Equal amounts of total proteins per sample were boiled in 10× SDS loading buffer for 10 min and separated by 8% or 10% SDS-PAGE gels. After transfer to polyvinylidene fluoride (PVDF) membranes, proteins were immunoblotted with primary antibodies followed by horseradish peroxidase-conjugated secondary antibody (CW0102S, CWBIO, 1:10,000). Signals were detected using a Chemiluminescence Imaging System (Biostep).

Co-IP assay

Protein extracts were incubated with GFP-Nanoab-agarose (GNA-50-1000, Lablead) for 2 hours at 4℃. Beads were washed three times with extraction buffer. After centrifugation at 1,000 g at 4℃ for 3 min, the precipitated proteins were eluted in 2× SDS loading buffer at 95℃ for 10 min. Proteins were subsequently separated by 10% SDS-PAGE gel and detected by immunoblotting with anti-GFP and anti-Myc antibodies.

In vivo ubiquitination assay

The conventional ubiquitination assay was performed as described (Fan et al., 2023). Briefly, six-day-old plants were treated with 100 μM MeJA and 50 μM Bortezomib (Btz) (S1013, Selleck) for 3 h and ground to a fine powder in liquid nitrogen. Total proteins were extracted with protein extraction buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (v/v) NP-40, 1% (v/v) Triton X-100, 1 mM PMSF, 1× complete protease inhibitor cocktail, 50 μM N-ethylmaleimide (CE5162, Coolaber), 50 μM MG132, 1× complete protease inhibitor cocktail]. The supernatants were centrifuged twice at 4℃, 13,000 g for 10 min. The resulting supernatants were incubated with pre-washed GFP-Nanoab-agarose for 3 h at 4℃, and washed three times with washing buffer [50 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.1% (v/v) NP-40, 1% (v/v) Triton X-100]. After centrifugation at 1,000 g, 4℃ for 3 min, the precipitated proteins were eluted in 2× SDS loading buffer at 95℃ for 10 min. Proteins were then separated by 10% SDS-PAGE gel and detected by immunoblotting with anti-GFP antibody and anti-UBQ11 rabbit polyclonal antibody (AS08307A, Agrisera).

Cell-free degradation assay

Cell-free degradation assays were performed as previously described (Xu et al., 2016). Total proteins extracts were prepared from six-day-old seedlings using extraction buffer [25 mM Tris-HCl (pH 7.5), 10 mM NaCl, 10 mM MgCl2, 5 mM PMSF, 5 mM DTT and 2 mM ATP]. The crude extract was then centrifuged at 13,000 g at 4℃ to remove debris, and the supernatants were adjusted to equal protein concentrations using the Bio-Rad assay. For purified JAZ1/JAZ3-His protein degradation, 20 μg recombinant JAZ1-His or JAZ3-His protein was incubated in 100 μL extract at 22℃ for 15, 30 and 60 min, then the reactions were stopped and the JAZ proteins were analyzed by immunoblotting using anti-His antibody (HT501; TransGen).

RNA-seq analysis

Col-0, myc2/3/4 and hy5-215 seeds were sown on MS medium supplemented with or without MeJA and grown under continuous white light at 22℃ or 28℃ for 6 days. Cotyledons were excised and harvested subsequently. Total RNA was extracted using an RNAprep Pure Plant Kit (Tiangen). Contaminating DNA was removed by digestion with RNase-free DNaseI (Tiangen) in accordance with the manufacturer’s instructions. Samples were submitted to Biomarker Biotechnology Corporation, Inc for sequencing. Three biological replicates were collected per genotype. Genes exhibiting a FC (Fold Change) ≥ 2 and FDR (False discovery rate) < 0.05 were identified as differentially expressed genes (DEGs) . The GO enrichment analysis was performed utilizing the online tool (https://david.ncifcrf.gov/conversion.jsp). GO terms with FDR < 0.05 were considered significantly enriched. GO term enrichment diagrams were generated using the website (https://www.chiplot.online/#BioPlot).

DAP-seq sampling and data analysis

DAP-seq was performed as previously described (O’Malley et al., 2016). Genomic DNA (gDNA) was extracted from the cotyledons of 6-day-old Col-0 seedlings grown on MS medium with 25 μM MeJA under continuous white light at 28℃ and purified by Bluescape Biotech (Hebei) and SeqHealth Tech (Wuhan). The fragmented gDNA was then used to construct libraries with the NEXTFLEX Rapid DNA-Seq Kit (PerkinElmer). The coding sequence of MYC2 and HY5 were cloned into a pFN19K HaloTag T7 SP6 Flexi vector and expressed using the TNT SP6 Coupled Wheat Germ Extract System (Promega). The expressed protein was purified and captured using Magne HaloTag Beads (Promega). The MYC2 and HY5-bound beads were incubated with adapter-ligated gDNA libraries. Eluted DNA was sequenced on an Illumina NovaSeq 6000 platform with two technical replicates. A negative control (Input) was prepared by incubating the gDNA libraries with beads in the absence of protein. The DAP-seq reads were aligned to the reference genome (TAIR https://www.arabidopsis.org). Peaks were called using MACS2, and the reproducibility of peaks between replicates was assessed using IDR (p < 0.05). MEME-CHIP was employed to identify conserved motifs within the peak regions, while Homer was used for peak annotation. GO enrichment analysis was performed utilizing the online tool (https://david.ncifcrf.gov/conversion.jsp). Target genes were defined as those with DAP-seq peaks located within 2 kb upstream of the ATG start codon.

ChIP-qPCR

ChIP assays were performed as described previously (Xu et al., 2024a). Briefly, plant tissues were crosslinked with 1% (v/v) formaldehyde under vacuum on ice for 15 min, and the reaction was quenched by adding 2 M glycine. Nuclei were isolated using lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, 0.1 mM PMSF, and 1× complete protease inhibitor cocktail). Chromatin was fragmented by sonication on ice to an average size of 250-500 bp. Immunoprecipitation was performed using anti-GFP antibody (D153-11, MBL) or anti-Myc antibody (M047-11, MBL), with goat anti-mouse IgG (HNM-25-1000, Lablead) as the negative control. Both input and ChIP samples were subjected to reverse crosslinking by incubation at 65℃ with Proteinase K overnight. DNA was subsequently purified by phenol extraction and recovered for analysis. qPCR was performed using Power SYBR Green Master Mix (Applied Biosystems) with gene-specific primers on immunoprecipitated DNA. Target gene promoter enrichment was presented as the percentage of input DNA, calculated by normalizing the immunoprecipitated DNA level against the input sample. ACTIN7 was used as reference genes, and primer sequences were listed in Supplementary Table 1.

Antibody production and information

Anti-HY5 (Jing et al., 2020) was produced in-house. Other antibodies were purchased commercially: anti-Actin (CW0264M, CWBio), anti-GFP (HT801, TransGen), anti-Myc (HT101, TransGen), anti-His (HT501, TransGen), anti-GST (ab184804, Abcam), anti-MYC2 (A20774, ABclonal), anti-RbcL (AS03037, Agrisera), anti-RbcS (AS07259, Agrisera), anti-HSP70 (M20041F, Abmart), anti-Lhcb3 (AS01002, Agrisera), anti-D1(AS05084, Agrisera), and anti-UBQ11(AS08307A, Agrisera).

Statistical analysis

All data analysis and visualization were performed using GraphPad Prism (version 10.6) and Origin 2019b software. The values are presented as mean standard deviation (SD). Statistical significance was determined by two-way analysis of variance (ANOVA) followed Tukey's multiple comparison test. Differences were considered statistically significant at p < 0.05, and distinct letters are used in the figures to denote significant differences between groups.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript addresses an interesting and timely topic, exploring how phyB integrates temperature and jasmonate (JA) signaling in regulating cotyledon yellowing. The study is comprehensive and provides a large set of phenotypic, molecular, and transcriptomic data, which highlights the authors’ effort and technical strength. The work is ambitious in scope and could make a valuable contribution to the field.\nHowever, the manuscript is also very complex and dense, making it challenging to read and interpret. The involvement of multiple genes, intricate pathways, and numerous experimental conditions often leaves the narrative confusing, and the genetic evidence supporting some of the key claims remains incomplete or requires deeper analysis. Below, I outline the major and minor concerns that should be addressed to improve clarity, strengthen the mechanistic conclusions, and ensure the data support the interpretations.\nMajor Concerns\n1. In Figure1 text, the author describe that “These results indicate that JA and high ambient temperature synergistically promote cotyledon yellowing.” “…their combination exerts a statistically validated additive effect.” These terms are contradictory. If the statistical interaction is additive, it should not be described as synergistic. Reanalyze the interaction term using two-way ANOVA and adjust terminology accordingly.\n2. In working model (Fig.9), the author argued that “…active phyB stabilizes JAZ … partially weakening JAZ’s suppression of MYC2 …” Stabilizing JAZ should strengthen, not weaken, MYC2 repression. This causal direction needs correction.\n3. In conclusion: the author wrote “…phyB … gates JA responses via direct COI1/JAZ interactions.” The manuscript provides evidence for phyB–JAZ interactions but no direct biochemical or genetic evidence for phyB–COI1 interaction, despite indicating such experiments in the Methods. The authors should provide direct biochemical or genetic evidence for a phyB–COI1 interaction, such as in vivo co-immunoprecipitation or in vitro pull-down assays under different temperature and JA conditions, to substantiate the claim of “direct COI1/JAZ interactions.”\n4. Multiple key figures rely on single-factor tests or uncorrected t-tests for factorial designs, whereas Fig. S7 uses two-way Fisher’s LSD. Two-way ANOVA with interaction terms and multiple-test correction should be applied consistently.\n5. Non-physiological Experimental Conditions\nSeveral key main-text figures, including Figure2A–B and Figure5, together with Supplementary Figures S6A–D and S8B–F,were performed using 100 μM MeJA combined with proteasome inhibitors (MG132 or Btz). While these conditions produce clear and stable degradation signals, they deviate from the 25 μM MeJA used for the phenotypic assays in Figure 1, Supplementary Figures S7-9.\nTo enhance physiological relevance and maintain consistency between biochemical and phenotypic data, it would be advisable to repeat or validate these degradation and ubiquitination assays under 25 μM JA ± 28 °C conditions. This adjustment would better align the mechanistic evidence with the biological responses observed in seedlings.\n6. Overgeneralization of Cotyledon Yellowing\nThe manuscript sometimes equates cotyledon yellowing with leaf senescence (Figure1 and related text), but chlorosis in developing cotyledons is not identical to true leaf senescence during later developmental stages. To improve accuracy, the authors could soften the wording to describe the phenotype as “chlorosis” or “early-stage yellowing,” or alternatively validate the claim by including assays in true leaves ,such as chlorophyll degradation profiles, senescence marker gene expression, or microscopy of mature leaf tissues — to demonstrate that similar senescence processes are occurring.\n7. Incomplete HY5/MYC2 Causal Evidence\nThe evidence for HY5 and MYC2 binding to downstream targets relies mainly on RNA-seq differential expression (Figure6 and FigureS13), DAP-seq binding profiles (FigureS15), and reporter assays (Figures 8E–F). While these data strongly suggest direct regulatory relationships, they remain in vitro or indirect. To strengthen this conclusion, in vivo evidence should be added, ChIP-qPCR or ChIP-seq to test binding of HY5 and MYC2 to promoters of key targets under 22 °C, 28 °C, and 28 °C + JA conditions to show dynamic occupancy consistent with the transcriptomic patterns.This addition would robustly validate the proposed HY5–MYC2 transcriptional regulatory network and close the current evidence gap between binding predictions and in vivo functional confirmation.\nMinor Issues\n1. Correct typographical errors such as “continues white light” → “continuous white light,” and standardize terminology throughout the figures.\n2. Clarify the normalization methods for chlorophyll, fluorescence, and ultrastructural measurements (e.g., per area or per fresh weight) to ensure reproducibility.\n3. Apply two-way ANOVA with interaction terms consistently for all factorial data, as noted above.\n4. The text in Figure 1 describes 28 °C treatment alone as causing a “mild pale phenotype”, while the gradient analysis in Supplementary Figure S1 shows almost no visible color difference up to 28 °C, with chlorosis only becoming evident at 30 °C. These descriptions are contradictory. Soften the main-text language to indicate that the 28 °C phenotype is subtle or variable, or provide quantitative data to reconcile the apparent inconsistency with the gradient assay.\n5. Many of the phenotypic and mechanistic assays, particularly those presented in Figure2, Figure4A–B, and Supplementary FiguresS3, S6, and S8 , primarily compare only two conditions: 22 °C (control) and 28 °C + JA. While these conditions highlight the combined effects of elevated temperature and JA treatment, this design limits the ability to distinguish independent temperature effects, independent JA effects, and their potential interaction. For example, in Figure4A–B, the JAZ stability and ubiquitination assays use only 22 °C + JA and 28 °C + JA, without including a 28 °C-only treatment to control for temperature-driven changes in JAZ turnover. Similarly, in the Figure 2 phenotype analyses, the lack of intermediate treatments or matched single-variable controls makes it difficult to precisely attribute the observed responses to temperature, JA, or their combination. To strengthen the mechanistic conclusions, it would be beneficial to expand the experimental design by including additional conditions, such as 28 °C alone and 22 °C + JA, across all phenotypic and biochemical assays. This would allow clearer interpretation of main effects versus interaction effects, improving the robustness of the data and ensuring consistency across figures and text.\n6. Some figure legends use inaccurate or inconsistent terms, such as “fluocam” (should be “FluorCam”). In addition, the RGB index used in Figure 1D and Figure 5E is not explained, making it difficult to interpret the direction of color change. Standardize terminology throughout and add a brief explanation of the RGB index in figure legends (e.g., whether higher values indicate more intense yellowing).\n7. Throughout the manuscript, cotyledon chlorosis in seedlings is repeatedly referred to as “leaf senescence.” However, cotyledon yellowing during early seedling development is not equivalent to true leaf senescence in mature tissues. Recommendation: Use more precise wording, such as “cotyledon chlorosis” or “early-stage yellowing,” unless additional assays are included in true leaves (e.g., chlorophyll degradation profiling, expression of canonical senescence markers, or ultrastructural analyses).", "reviewer_2": "This manuscript, which aims to elucidate the molecular-level crosstalk between JA signaling and high ambient temperature in Arabidopsis thaliana, was reviewed with considerable interest. The authors have assembled an impressive body of data, and the proposed model involving the interaction between PhyB and JAZ1/3 is both novel and of potential significance. The study thus has the potential to contribute meaningfully to our understanding of this signaling network.\nThat said, the manuscript would benefit from more careful presentation and interpretation of the experimental results. First, there is a notable imbalance between the comprehensive physiological analyses in the first half and the mechanistic experiments in the latter half, which lack sufficient depth and clarity. The sections on HY5 and MYC2, while supported by numerous experiments, remain less well integrated into the overall model compared to the PhyB–JAZ module, leaving this part of the study somewhat disconnected.\nSecond, the presentation and interpretation of experimental results require improvement. In several cases—particularly in the supplementary figures—the explanations are insufficient, and certain results appear internally inconsistent. These issues reduce both the clarity and reproducibility of the study. More careful presentation, together with additional clarification and, where necessary, refinement or repetition of specific experiments, would substantially strengthen the manuscript.\nFinally, although the interaction between high ambient temperature and JA signaling has been previously reported, the main significance and novelty of the present work should lie in the detailed elucidation of the underlying molecular mechanisms. The current insufficiency in mechanistic depth, together with the concerns noted above, represents a major weakness.\nIn summary, while this manuscript presents a large dataset and raises interesting possibilities, substantial revisions are required to improve data presentation, ensure consistency, and more clearly establish the mechanistic framework before the work can be considered for publication.\nMajor Comments\n• Line 143: The authors describe the effect as “synergistic.” However, based on the data, the influence of JA appears dominant, and the effect seems additive rather than synergistic. A more cautious wording such as “additive” may be more appropriate.\n• Line 158: The choice of 28 °C as the high-temperature condition is not well justified. Figure S1C suggests that 30 °C may be a more suitable condition. Moreover, the data in Fig. 1C and Fig. S1C appear inconsistent for the 28 °C + JA chlorophyll content. Please check and, if necessary, replace the data.\n• Fig. S1. To properly address the function of PhyB, experiments under both red and far-red light conditions are necessary.\n• Fig. 2A, B and lines 192–195. The authors describe the COI1-OX line as showing the opposite phenotype to coi1-8; however, the chlorophyll content data do not appear to support a clear difference. This ambiguity may stem from the sentence structure, which makes the authors' intention unclear. Furthermore, the conclusion in line 201 seems valid only for the 28°C treatment, but not for the 22°C + JA and 28°C + JA conditions. This discrepancy, if not a misunderstanding on my part, should be addressed in the Discussion.\n• Line 198: In phyB-9, chlorophyll content is already reduced under 28 °C treatment alone, and in 28 °C + JA treatment the chlorophyll content even appears increased. If this interpretation is correct, an explanation of this phenomenon is necessary.\n• Line 201, 213: The relationship between phyB and COI1 is described as “antagonistic.” However, at this stage it is unclear whether these factors act cooperatively or oppositely. A more neutral term such as “opposing role” would be preferable.\n• Fig. 2C. The differences between 22°C and 28°C, particularly for JAZ1, are not presented quantitatively. I recommend that the authors conduct at least three independent experiments and present the corresponding quantitative data.\n• Fig. 2 and L224-. The authors should clarify their rationale for selecting JAZ1 and JAZ3 for analysis from the JAZ gene family, as other members, such as JAZ10, may also have important functions.\n• Line 230: The authors report that full-length phyB does not interact with JAZ, whereas the C-terminal domain does. The rationale for this difference should be discussed. In addition, the choice of truncation constructs (C451–, C651–) should be justified with reference to their functional domains.\n• Line 240: The statement “reduced phyB levels” appears inconsistent with Fig. 3D, where phyB protein levels in the input do not obviously differ. Please clarify.\n• Line 249: The statement that reduced chlorophyll levels in phyB mutants are “likely due to diminished cotyledon area” is not supported by data. Since chlorophyll content is expressed per g FW, changes in area would not necessarily alter chlorophyll concentration. Please provide area measurements or revise the explanation.\n• Line 268: In Fig. 4B, the authors describe ubiquitinated JAZ1 levels in phyB-9 as “not significant.” However, they appear clearly elevated compared to WT. More quantitative data should be provided.\n• Line 283 (Fig. 4D): In phyB mutants, JAZ degradation still occurs in the presence of MG132, suggesting an additional proteolytic pathway beyond the proteasome. While other factors are briefly mentioned in the Discussion, the possibility of non-proteasomal degradation should also be explicitly discussed.\n• Line 297: The statement that “phyB mutant exhibited ~50% lower JAZ levels” is not consistently supported across all data (e.g., Figs. 4F/G appear comparable to WT). Presumably, values are normalized to 0 h = 1.0, but this should be stated in the figure legend. Moreover, quantification sometimes appears inconsistent with band intensities. Please re-examine and clarify.\n• Fig. 2C. The differences between 22°C and 28°C, particularly for JAZ1, are not presented quantitatively. I recommend that the authors conduct at least three independent experiments and present the corresponding quantitative data.\n• Fig. 4A and 4B. Control samples lacking MeJA treatment are missing. Although the ubiquitin bands may be faint, they should still be shown. Moreover, the rationale for using the inhibitor Bortezomib (Btz) should be explained in the Results, as it likely inhibited protein degradation and may have obscured the visualization of the ubiquitin bands.\n• L398–L556. The description of the RNA-seq results is excessively long and should be substantially shortened. The section beginning at line 398 merely lists the number of genes, which is uninformative. The authors must focus on the most essential findings and present this section concisely. The section starting at line 508 has the same issue and should also be condensed.\n• Fig. 8A, B. Given that a genome-wide analysis of HY5 and MYC2 is already presented in Fig. 7, the rationale for performing the Y1H assay is unclear and should be explicitly justified.\n• Fig. 8C–F. The reporter assays were performed only in the wild-type background, which is insufficient. The authors should also use COI1 and phyB transgenic or mutant lines to assess the specific contributions of these factors. Furthermore, it is a significant omission that the effects of temperature and MeJA—central to this study—are not addressed in these results.\n• Fig. 9. The proposed model is difficult to interpret. To improve clarity, it should be redrawn to separately illustrate the scenarios with and without JA under different temperature conditions.\nMinor Comments\n• L54. The term \"Jasmonates\" is generally used.\n• L62. Please change \"hormone pathway\" to \"hormone signaling pathway.\"\n• L78 and L105. Please replace \"Beyond\" with \"In addition to.\"\n• L124. Please include the relevant citation(s) and elaborate on the biochemical characteristics and functional significance of 12-HSO₄-JA.\n• L145. Please remove the sentence \"JA and high…,\" as this point was already made in the Introduction.\n• L158. Please change \"selected\" to \"focused on.\"\n• L160 and later. Please correct the formatting to remove the space between the number and the °C symbol (e.g., 22°C).\n• Line 166: The phrase “a dramatic reduction in area” appears exaggerated given the data in Fig. 1B. Please moderate the description.\n• L168. Please rephrase \"temperature and JA application independently\" to \"either elevated temperature or JA application alone.\"\n• L169. Please rephrase \"additive\" to \"additive or synergistic.\"\n• Figures 1C and 1F. Please use ANOVA followed by a two-sided Fisher’s LSD test, as comparisons need to be made across all samples.\n• L261. Please remove \"firstly.\"\n• L276. Please clarify what is meant by \"either directly or indirectly.\"\n• Fig. S6E–H. The rationale for these experiments is unclear. Specifically, it should be explained why in vitro experiments were necessary in addition to the in vivo analyses. This point should be described at line 301.\n• L441. Please change \"new\" to \"other.\"\n• Throughout the manuscript, the term “JA” is frequently used, but the treatment is specifically MeJA. Please revise for accuracy.\n• Figure 1E: Treatment conditions (temperature, JA treatment) are not indicated. Please add.\n• Figures S1 and S2: The number of biological replicates (n) is missing. Please include this throughout the manuscript.\n• Figure S3A: Cotyledon area data for 28 °C and 22 °C + MeJA conditions should be added.\n• Figure S4F: The 22 °C + JA treatment for 35S:JAZ3-GFP lacks statistical analysis. Please include.", "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."}, "author_response": "Response to reviewer comments\nReviewer #1 (Remarks to the Author):\nThis manuscript addresses an interesting and timely topic, exploring how phyB\nintegrates temperature and jasmonate (JA) signaling in regulating cotyledon yellowing.\nThe study is comprehensive and provides a large set of phenotypic, molecular, and\ntranscriptomic data, which highlights the authors’ effort and technical strength. The\nwork is ambitious in scope and could make a valuable contribution to the field.\nHowever, the manuscript is also very complex and dense, making it challenging to read\nand interpret. The involvement of multiple genes, intricate pathways, and numerous\nexperimental conditions often leaves the narrative confusing, and the genetic evidence\nsupporting some of the key claims remains incomplete or requires deeper analysis.\nBelow, I outline the major and minor concerns that should be addressed to improve\nclarity, strengthen the mechanistic conclusions, and ensure the data support the\ninterpretations.\nMajor Concerns\n1. In Figure1 text, the author describes that “These results indicate that JA and high\nambient temperature synergistically promote cotyledon yellowing.” “…their\ncombination exerts a statistically validated additive effect.” These terms are\ncontradictory. If the statistical interaction is additive, it should not be described as\nsynergistic. Reanalyze the interaction term using two-way ANOVA and adjust\nterminology accordingly.\nResponse：We sincerely thank the reviewer for this critical observation and for pointing\nout the contradictory terminology. We have reanalyzed the data for Fig.1 using a two-\nway ANOVA to formally test the interaction between JA treatment and high\ntemperature. The statistical analysis confirmed that the interaction was significant,\nindicating that the combined effect of JA and high temperature is indeed synergistic,\nnot additive. We have revised the text.\n1\n2. In working model (Fig.9), the author argued that “…active phyB stabilizes JAZ …\npartially weakening JAZ’s suppression of MYC2 …” Stabilizing JAZ should\nstrengthen, not weaken, MYC2 repression. This causal direction needs correction.\nResponse：Thank you for your important comment. We agree that the original\ndescription of how active phyB influences JAZ-MYC2 signaling was imprecise and\ncould be misleading. As suggested, we have carefully re-evaluated the causal direction\nin our model. Accordingly, we have revised the model diagram (Fig. 9) and updated\nthe relevant text (line 1606-1621) to reflect the proposed relationship more accurately.\nThe correction ensures that the narrative aligns logically with the established signaling\nlogic. We appreciate your valuable insight, which has been crucial in enhancing the\nclarity and accuracy of our working model.\n3. In conclusion: the author wrote “…phyB … gates JA responses via direct COI1/JAZ\ninteractions.” The manuscript provides evidence for phyB–JAZ interactions but no\ndirect biochemical or genetic evidence for phyB–COI1 interaction, despite indicating\nsuch experiments in the Methods. The authors should provide direct biochemical or\ngenetic evidence for a phyB–COI1 interaction, such as in vivo co-immunoprecipitation\nor in vitro pull-down assays under different temperature and JA conditions, to\nsubstantiate the claim of “direct COI1/JAZ interactions.”\nResponse：Thanks for your suggestion. We are sorry for the mis-interpretation about\nphyB-COI1 interaction. We have revised the statement as “our study elucidates a\nhierarchical signaling network where phyB senses temperature and light to gate JA\nresponses via JAZs, HY5 and MYC2 proteins.”\n4. Multiple key figures rely on single-factor tests or uncorrected t-tests for factorial\ndesigns, whereas Fig. S7 uses two-way Fisher’s LSD. Two-way ANOVA with\ninteraction terms and multiple-test correction should be applied consistently.\nResponse：Thanks for your suggestion. We have now consistently applied a two-way\nANOVA with interaction terms, followed by appropriate multiple-test corrections\nacross all relevant analyses. The updated statistical approach has been implemented in\nthe revised Supplementary Fig. 8C (previously Fig. S7C), where detailed results are\n2\nprovided.\n5. Non-physiological Experimental Conditions\nSeveral key main-text figures, including Figure2A–B and Figure5, together with\nSupplementary Figures S6A–D and S8B–F, were performed using 100 μM MeJA\ncombined with proteasome inhibitors (MG132 or Btz). While these conditions produce\nclear and stable degradation signals, they deviate from the 25 μM MeJA used for the\nphenotypic assays in Figure 1, Supplementary Figures S7-9.\nTo enhance physiological relevance and maintain consistency between biochemical and\nphenotypic data, it would be advisable to repeat or validate these degradation and\nubiquitination assays under 25 μM JA ± 28°C conditions. This adjustment would better\nalign the mechanistic evidence with the biological responses observed in seedlings.\nResponse：We thank the reviewer for highlighting the need for consistency in MeJA\nconcentrations across our experiments. We fully agree that aligning biochemical and\nphenotypic conditions is crucial for physiological relevance. Following the reviewer's\nsuggestion, we initially performed the degradation assay under physiological conditions\n(28°C+25 μM MeJA). The results (Supplementary Fig. 6E, F) demonstrate a consistent\ndegradation trend mirroring the 100 μM MeJA response, despite with slightly slower\nkinetics. However, for the subsequent, more demanding biochemical experiments (such\nas ubiquitination assays), we found that the signal intensity under 25 μM MeJA was\noften at the lower limit of reliable detection for quantitative comparisons. The 100 μM\nMeJA condition provided a more robust and reproducible signal, which was essential\nfor definitively establishing the degradation mechanism. Given the qualitative\nconsistency between the 25 μM and 100 μM treatments confirmed in Supplementary\nFig. 6, we are confident that the molecular mechanisms uncovered using the higher\nconcentration are operative under physiological stress. Therefore, we employed the 100\nμM condition as a validated and practical experimental condition to dissect the pathway\nin detail. Meanwhile, it has been widely used in previous studies that a low\nconcentration of chemical treatment in physiological experiments while a high\nconcentration with short-term treatment for degradation assays.\n3\n6. Over generalization of Cotyledon Yellowing\nThe manuscript sometimes equates cotyledon yellowing with leaf senescence (Figure1\nand related text), but chlorosis in developing cotyledons is not identical to true leaf\nsenescence during later developmental stages. To improve accuracy, the authors could\nsoften the wording to describe the phenotype as “chlorosis” or “early-stage yellowing,”\nor alternatively validate the claim by including assays in true leaves, such as chlorophyll\ndegradation profiles, senescence marker gene expression, or microscopy of mature leaf\ntissues — to demonstrate that similar senescence processes are occurring.\nResponse：We thank the reviewer for this insightful comment regarding the precise\nterminology used to describe the observed phenotype. We agree that directly equating\ncotyledon yellowing with leaf senescence in mature tissues is overly simplistic.\nFollowing the reviewer's advice, we have revised our manuscript to improve descriptive\naccuracy. Throughout the manuscript, particularly in the text related to Figure 1, we\nhave softened our language, consistently referring to the cotyledon phenotype as\n“yellowing” or “chlorosis” rather than “senescence”. This change more accurately\ndescribes the visible phenotype without making definitive claims about the\ndevelopmental process in these specific organs.\n7. Incomplete HY5/MYC2 Causal Evidence\nThe evidence for HY5 and MYC2 binding to downstream targets relies mainly on\nRNA-seq differential expression (Figure 6 and Figure S13), DAP-seq binding profiles\n(Figure S15), and reporter assays (Figures 8E–F). While these data strongly suggest\n4\ndirect regulatory relationships, they remain in vitro or indirect. To strengthen this\nconclusion, in vivo evidence should be added, ChIP-qPCR or ChIP-seq to test binding\nof HY5 and MYC2 to promoters of key targets under 22°C, 28°C, and 28°C + JA\nconditions to show dynamic occupancy consistent with the transcriptomic patterns.\nThis addition would robustly validate the proposed HY5–MYC2 transcriptional\nregulatory network and close the current evidence gap between binding predictions and\nin vivo functional confirmation.\nResponse：We sincerely thank the reviewer for this insightful comment regarding the\nneed for in vivo evidence to solidify the direct regulatory relationships between\nHY5/MYC2 and their downstream targets. We agree that combining in vitro DAP-seq\ndata with in vivo validation is crucial for establishing the proposed transcriptional\nnetwork. In direct response to this suggestion, we have performed Chromatin\nImmunoprecipitation quantitative PCR (ChIP-qPCR) assays to test the in vivo binding\nof HY5 and MYC2 to the promoters of key target genes under the critical experimental\nconditions: 22°C, 28°C, and 28°C + JA. The new results (Line 555-570), presented in\nthe revised Fig. 8A-D and Supplementary Fig. 18A-D, demonstrate dynamic occupancy\nby both transcription factors, which align fully with our transcriptomic data.\nSpecifically, the ChIP-qPCR data confirm that the binding enrichment of HY5 and\nMYC2 at their target promoters changes significantly across the different temperature\nand JA treatments, mirroring the expression changes observed in our RNA-seq data.\nThis new in vivo evidence directly bridges the gap between our initial binding\npredictions and functional confirmation, thereby strongly validating the core of the\nproposed HY5-MYC2 regulatory module. We believe these additions have\nsignificantly strengthened the conclusions of our study.\n1. Correct typographical errors such as “continues white light” → “continuous white\nlight,” and standardize terminology throughout the figures.\nResponse：We have corrected these errors throughout the figures.\n2. Clarify the normalization methods for chlorophyll, fluorescence, and ultrastructural\nmeasurements (e.g., per area or per fresh weight) to ensure reproducibility.\nResponse：We thank the reviewer for this important suggestion to improve the clarity\nand reproducibility of our methodologies. We have explicitly stated the normalization\n6\nmethods for all relevant measurements in the revised Materials and Methods section.\n3. Apply two-way ANOVA with interaction terms consistently for all factorial data,\nas noted above.\nResponse：We agree with the reviewer on the need for consistent statistical\nmethodology. Accordingly, we have applied a two-way ANOVA with interaction terms\nfollowed by appropriate multiple-test corrections for all factorial experimental data\nthroughout the manuscript, and the results have been updated accordingly.\n4. The text in Figure 1 describes 28°C treatment alone as causing a “mild pale\nphenotype”, while the gradient analysis in Supplementary Figure S1 shows almost no\nvisible color difference up to 28°C, with chlorosis only becoming evident at 30°C.\nThese descriptions are contradictory. Soften the main-text language to indicate that the\n28°C phenotype is subtle or variable or provide quantitative data to reconcile the\napparent inconsistency with the gradient assay.\nResponse：We thank the reviewer for this excellent observation regarding the apparent\ninconsistency between the text description of the 28°C phenotype and the gradient assay\nin Supplementary Fig.1 We have revised the original description from \"...whereas 28°C\ntreatment alone caused a mild pale phenotype...\" to \"...while the phenotype induced by\n28°C alone was subtle and variable...\".\n5. Many of the phenotypic and mechanistic assays, particularly those presented in\nFigure 2, Figure 4A–B, and Supplementary FiguresS3, S6, and S8, primarily compare\nonly two conditions: 22°C (control) and 28°C + JA. While these conditions highlight\nthe combined effects of elevated temperature and JA treatment, this design limits the\nability to distinguish independent temperature effects, independent JA effects, and their\npotential interaction. For example, in Figure4A–B, the JAZ stability and ubiquitination\nassays use only 22°C + JA and 28°C + JA, without including a 28°C-only treatment to\ncontrol for temperature-driven changes in JAZ turnover. Similarly, in the Figure 2\nphenotype analyses, the lack of intermediate treatments or matched single-variable\ncontrols makes it difficult to precisely attribute the observed responses to temperature,\nbeneficial to expand the experimental design by including additional conditions, such\nas 28°C alone and 22°C + JA, across all phenotypic and biochemical assays. This would\nallow clearer interpretation of main effects versus interaction effects, improving the\nrobustness of the data and ensuring consistency across figures and text.\nResponse：We sincerely thank the reviewer for this critical and constructive comment\nregarding our experimental design. To address this point thoroughly and substantially\nstrengthen our mechanistic conclusions, we have performed a comprehensive set of\nnew experiments. Specifically, as suggested, we have now included the 28°C alone and\n22°C + JA conditions across all key phenotypic and biochemical assays.\nThe changes we have made are detailed below:\nPhenotype Analyses (Related to original Fig. 2):\nWe have supplemented the statistical data on the phenotypes for the 28°C alone\ntreatment and the 22°C + JA control group. These new data, presented in the revised\nFig. 2 and Supplementary Fig.3, enable us to attribute the observed phenotypes more\nclearly. The results demonstrate that high temperature alone induces moderate\nyellowing, JA alone has a mild effect, but their combination produces a strong,\nsynergistic enhancement. We have updated the text and statistical analyses (using two-\nway ANOVA analysis) to reflect these new findings.\nJAZ Stability and Ubiquitination Assays (Related to original Fig. 4A-B):\nWe have re-examined the experiments on JAZ protein degradation and found that\nelevated temperature alone increases JAZ protein abundance (Supplementary Fig. 6A-\nD), as previously described1. Following the reviewer's suggestion, we initially\nperformed the degradation assay under physiological conditions (28°C+25 μM MeJA).\nThe results (Supplementary Fig. 6E, F) demonstrate a consistent degradation trend\nmirroring the 100 μM MeJA response, despite with slightly slower kinetics. However,\nfor the subsequent, more sensitive biochemical assays—such as ubiquitination analysis\nand co-treatment with proteasome inhibitors—we found that the signal intensity under\n25 μM MeJA was often near the lower detection limit, making reliable quantitative\ncomparisons challenging. For this reason, we were unable to obtain clear and\nreproducible data to evaluate the effect of phyB on JAZ ubiquitination under long-term\n8\nhigh-temperature and JA co-treatment within the current experimental framework.\nWe fully acknowledge the importance of this point and plan to further optimize the\nassay conditions—for example, by adjusting detection methods or sample processing\nprotocols—to address this technical limitation in future studies. We hope to revisit this\nquestion with more robust methodology and appreciate the reviewer’s understanding.\n6. Some figure legends use inaccurate or inconsistent terms, such as “fluocam” (should\nbe “FluorCam”). In addition, the RGB index used in Figure 1D and Figure 5E is not\nexplained, making it difficult to interpret the direction of color change. Standardize\nterminology throughout and add a brief explanation of the RGB index in figure legends\n(e.g., whether higher values indicate more intense yellowing).\nResponse：Thanks for your advice. We have corrected the incorrect terminology in the\narticle and added an explanation regarding the RGB index. The revised text now\nexplicitly states: “The RGB index (calculated as the Red/Green ratio) is used as a\nquantitative measure of leaf yellowing, where higher values indicate more intense\nyellowing.”\n6. Throughout the manuscript, cotyledon chlorosis in seedlings is repeatedly\nreferred to as “leaf senescence.” However, cotyledon yellowing during early seedling\ndevelopment is not equivalent to true leaf senescence in mature tissues.\nRecommendation: Use more precise wording, such as “cotyledon chlorosis” or “early-\nstage yellowing,” unless additional assays are included in true leaves (e.g., chlorophyll\ndegradation profiling, expression of canonical senescence markers, or ultrastructural\nanalyses).\nResponse：Thanks for your advice. Accordingly, we have replaced the term “leaf\nsenescence” with “cotyledon chlorosis” throughout the manuscript. We have updated\nthe relevant Results and Discussion sections accordingly and believe these revisions\nhave significantly improved the accuracy and rigor of our manuscript.\nThis manuscript, which aims to elucidate the molecular-level crosstalk between JA\nsignaling and high ambient temperature in Arabidopsis thaliana, was reviewed with\nconsiderable interest. The authors have assembled an impressive body of data, and the\nproposed model involving the interaction between PhyB and JAZ1/3 is both novel and\nof potential significance. The study thus has the potential to contribute meaningfully to\nour understanding of this signaling network.\nThat said, the manuscript would benefit from more careful presentation and\ninterpretation of the experimental results. First, there is a notable imbalance between\nthe comprehensive physiological analyses in the first half and the mechanistic\nexperiments in the latter half, which lack sufficient depth and clarity. The sections on\nHY5 and MYC2, while supported by numerous experiments, remain less well\nintegrated into the overall model compared to the PhyB–JAZ module, leaving this part\nof the study somewhat disconnected.\nSecond, the presentation and interpretation of experimental results require\nimprovement. In several cases—particularly in the supplementary figures—the\nexplanations are insufficient, and certain results appear internally inconsistent. These\nissues reduce both the clarity and reproducibility of the study. More careful presentation,\ntogether with additional clarification and, where necessary, refinement or repetition of\nspecific experiments, would substantially strengthen the manuscript.\nFinally, although the interaction between high ambient temperature and JA\nsignaling has been previously reported, the main significance and novelty of the present\nwork should lie in the detailed elucidation of the underlying molecular mechanisms.\nThe current insufficiency in mechanistic depth, together with the concerns noted above,\nrepresents a major weakness.\nIn summary, while this manuscript presents a large dataset and raises interesting\npossibilities, substantial revisions are required to improve data presentation, ensure\nconsistency, and more clearly establish the mechanistic framework before the work can\nbe considered for publication.\nResponse：We are grateful for this reviewer’s thoughtful comments and suggestions\n10\non our presentation and interpretation. Additional experiments were performed and the\ndetailed responses are shown below.\nMajor Comments\n1. Line 143: The authors describe the effect as “synergistic.” However, based on the\ndata, the influence of JA appears dominant, and the effect seems additive rather than\nsynergistic. A more cautious wording such as “additive” may be more appropriate.\nResponse：We thank the reviewer for raising this important point regarding the\ninterpretation of the JA and high-temperature interaction. We agree that rigorously\ndistinguishing between synergistic and additive effects requires formal statistical\ntesting. As also suggested by the first reviewer, we have performed a two-way ANOVA\nwith an interaction term to formally assess the relationship between JA treatment and\ntemperature for the data presented in Fig.1. The analysis revealed a statistically\nsignificant interaction (p < 0.05). Based on this objective statistical evidence, we have\nretained the term \"synergistic\" in the revised manuscript (Line 138). We are grateful\nthat the reviewer's comment, which prompted this more rigorous analysis, which we\nbelieve has strengthened the support for our initial conclusion.\n2. Line 158: The choice of 28 °C as the high-temperature condition is not well justified.\nFigure S1C suggests that 30 °C may be a more suitable condition. Moreover, the data\nin Fig. 1C and Fig. S1C appear inconsistent for the 28°C + JA chlorophyll content.\nPlease check and, if necessary, replace the data.\nResponse：Based on our experimental observations and considerations within the\nresearch context, we selected 28℃ as the high-temperature condition for the following\nreasons: although 30℃ did induce a more pronounced phenotype, it approaches the\ncritical threshold for activating classical heat stress responses in plants. Since our study\nfocuses on warm temperature rather than heat stress, we deliberately chose 28℃ based\non our temperature gradient experiments. This temperature has been well-established\nin the thermomorphogenesis field, as it effectively induces relevant response while\nminimizing confounding activation of heat stress pathways. This selection ensures our\n11\nfindings are directly comparable to the existing literatures.\nRegarding the chlorophyll content data, we have verified our experimental results\nand confirmed the consistency. The observed variation between different figures may\narise from different experimental batches, but all presented data are reliable and\nreproducible. We appreciate the reviewer's insightful comment and hope this\nclarification adequately addresses the concerns.\n3. Fig. S1. To properly address the function of PhyB, experiments under both red and\nfar-red light conditions are necessary.\nResponse：Thank you for this insightful comment. As you rightly pointed out, we have\nnow conducted the necessary experiments under both red light (R) and far-red light (FR)\nconditions to properly assess phyB’s function. The new data (Supplementary Fig.3E-\nH) clearly show that the phenotypic defects of the phyB-9 mutants are strongly\nmanifested under red light but absent under far-red light. This result is critical, as it\nprovides direct genetic evidence that phyB is the primary photoreceptor mediating red\nlight signaling in this process. The lack of a phenotype under far-red light confirms that\nthe observed effects are specific to the loss of phyB function and not a general\nphotomorphogenic defect. We thank the reviewer for prompting this important\nclarification, which has strengthened our study.\n12\n4. Fig. 2A, B and lines 192–195. The authors describe the COI1-OX line as showing\nthe opposite phenotype to coi1-8; however, the chlorophyll content data do not appear\nto support a clear difference. This ambiguity may stem from the sentence structure,\nwhich makes the authors' intention unclear. Furthermore, the conclusion in line 201\nseems valid only for the 28°C treatment, but not for the 22°C + JA and 28°C + JA\nconditions. This discrepancy, if not a misunderstanding on my part, should be addressed\nin the Discussion.\nResponse：We thank the reviewer for these insightful comments regarding the clarity\nof our description and the scope of our conclusions. We have addressed both points\ncarefully.\nFirst, to improve clarity: our intention was to state that under specific conditions\n(28°C+JA), the 35S:COI1-GFP line showed a trend opposite to coi1-8, particularly in\nthe chlorophyll degradation phenotype, although the difference was not always\nstatistically significant in the chlorophyll assay. To reflect this more accurately, we\nhave revised the text (Lines 197-200) to “under 28℃ + MeJA, the 35S: COI1-GFP line\ntended to have elevated chlorophyll loss compared with the wild-type, which contrasted\n13\nwith the impaired chlorophyll loss observed in the coi1-8 mutant” All statements in the\nrevised text are now strictly supported by the statistical analyses presented in the\nupdated Fig. 2A, B.\nSecond, to address the overgeneralization of our conclusions，we have added a\nnew paragraph to the Discussion (Line 670-687). There, we explicitly discuss the\ncomplex interaction between JA signaling and warm temperature. We note that\nalthough COI1 is necessary for the synergistic response, the application of exogenous\nJA under our experimental conditions may have saturated or engaged additional\nsignaling pathways that modulated the phenotypic outcome. This helps explain why a\nsimple opposite phenotype was not consistently observed across all JA-treatment\nconditions.\nWe believe these revisions have substantially improved the clarity and precision\nof our manuscript, and we thank the reviewer once again for the valuable feedback.\n5. Line 198: In phyB-9, chlorophyll content is already reduced under 28 °C treatment\nalone, and in 28 °C + JA treatment the chlorophyll content even appears increased. If\nthis interpretation is correct, an explanation of this phenomenon is necessary.\nResponse：We thank the reviewer for this astute observation regarding the chlorophyll\ncontent in the phyB-9 mutant. The reviewer is correct that under the 28°C+ JA condition,\nthe measured chlorophyll content per fresh weight appears elevated compared to the\n28°C treatment alone. We have carefully investigated this phenomenon and propose a\nplausible explanation based on the morphological differences between the treatments.\nAs now shown in the new Supplementary Fig. 3A, the cotyledon area of phyB-9\nseedlings at 28°C is significantly smaller than that of seedlings treated with 28°C +\nMeJA. Since our chlorophyll measurement was normalized to the fresh weight of a\nfixed number of seedlings (20), this normalization method can be sensitive to\ndevelopmental variation. We hypothesize that JA application, while not restoring the\nchlorophyll concentration to wild-type levels, may partially alleviate the severe\nmorphological impairment caused by high temperature in the phyB-9 background. This\nresults in seedlings with larger cotyledons and thus a higher total chlorophyll content\nper seedling. When a fixed number of seedlings is pooled for measurement, this\n14\ndevelopmental difference manifests as an increase in chlorophyll per fresh weight in\nthe 28°C + MeJA group. This interpretation is consistent with our overall model that\nJA signaling interacts with the thermo-morphogenic pathway, highlighting JA's\ncomplex role in modulating both chlorophyll metabolism and morphological responses\nto temperature. We have added a sentence in the manuscript (Line 195-196) to\nacknowledge and briefly explain this observation to avoid confusion.\n6. Line 201, 213: The relationship between phyB and COI1 is described as\n“antagonistic.” However, at this stage it is unclear whether these factors act\ncooperatively or oppositely. A more neutral term such as “opposing role” would be\npreferable.\nResponse：Thanks for your suggestion. We changed the “antagonistic” to “opposite”.\n7. Fig. 2C. The differences between 22°C and 28°C, particularly for JAZ1, are not\npresented quantitatively. I recommend that the authors conduct at least three\nindependent experiments and present the corresponding quantitative data.\nResponse: We thank the reviewer for this critical suggestion regarding the quantitative\npresentation of the data in the original Fig. 3D. We agree that robust quantification and\nstatistical analysis are essential. We conducted two biological replicates, both of which\nyielded consistent experimental results confirming that high temperature indeed\nweakens the interaction between phyB and JAZs. To ensure rigor, we performed\ngrayscale analysis, as now presented in the revised Fig. 3D, the quantitative data with\nstatistical analysis further validated our conclusion.\n8. Fig. 2 and L224-. The authors should clarify their rationale for selecting JAZ1 and\nJAZ3 for analysis from the JAZ gene family, as other members, such as JAZ10, may\nalso have important functions.\nResponse: We thank the reviewer for raising this point. Our selection of JAZ1 and\nJAZ3 was based on their well-characterized roles as central repressors in the JA\npathway and their robust, representative expression changes in our preliminary data2.\nWe acknowledge that other members like JAZ10 are functionally important, and their\n15\ninvestigation in the context of thermomorphogenesis represents a valuable future\ndirection.\n9. Line 230: The authors report that full-length phyB does not interact with JAZ,\nwhereas the C-terminal domain does. The rationale for this difference should be\ndiscussed. In addition, the choice of truncation constructs (C451–, C651–) should be\njustified with reference to their functional domains.\nResponse：In the Y2H assay, the full-length phyB did not interact with JAZ, whereas\nits C-terminal domain did. However, both LCI and BiFC assays confirmed their\ninteraction in planta. There may be several reasons for this. First, the full-length phyB\nmay contain an autoinhibitory domain that restrains its interaction capability; removal\nof this domain in the truncated form may permit JAZ binding. Second, the large size\nand structural complexity of full-length phyB could sterically hinder access to the JAZ\ninteraction interface. Third, the full-length protein may adopt a more stable, rigid\nconformations that masks the interaction site, while the truncated version may exhibit\nenhanced flexibility, facilitating a transition to an active binding state. Finally, the plant\ncellular environment may provide essential co-factors or conditions required for the\nfull-length phyB-JAZ interaction, which are absent in simplified yeast systems. We\nhave expanded the discussion of these possibilities with relevant references to known\nfunctional domains in the revised manuscript (Line 240-244).\n10. Line 240: The statement “reduced phyB levels” appears inconsistent with Fig. 3D,\nwhere phyB protein levels in the input do not obviously differ. Please clarify.\nResponse：We thank the reviewer for rightly noting that the phyB protein levels in the\nInput samples of Fig. 3D do not appear visually distinct across temperature treatments.\nIn response, we have conducted grayscale quantification of the phyB bands from the\nInput lanes. The results demonstrate a significant reduction in phyB levels following\nhigh-temperature treatment. We have updated the figure for Fig. 3D to include these\nquantitative results and amended the text at line 250-251 to describe the observed\nchange more precisely. Accordingly, the statement “reduced phyB levels” is now fully\nconsistent with the quantified experimental data.\n16\n11. Line 249: The statement that reduced chlorophyll levels in phyB mutants are “likely\ndue to diminished cotyledon area” is not supported by data. Since chlorophyll content\nis expressed per g FW, changes in area would not necessarily alter chlorophyll\nconcentration. Please provide area measurements or revise the explanation.\nResponse：We have carefully investigated this phenomenon and propose a plausible\nexplanation rooted in the morphological differences between the treatments. As shown\nin the new Supplementary Fig. 3A, the cotyledon area of phyB-9 seedlings at 28°C is\nsignificantly smaller than that of seedlings treated with 28°C+JA. Since our chlorophyll\nmeasurement was normalized to the fresh weight of a fixed number of seedlings (20),\nthis normalization method can be sensitive to developmental variation. The decrease in\ncotyledon area will reduce the proportion of photosynthetic tissue per seedling, which\ncan consequently affect the measured chlorophyll content.\n12. Line 268: In Fig. 4B, the authors describe ubiquitinated JAZ1 levels in phyB-9 as\n“not significant.” However, they appear clearly elevated compared to WT. More\nquantitative data should be provided.\nResponse：We thank the reviewer for this insightful observation. To address this point\ndefinitively, we have repeated the in vivo ubiquitination assay. The new results, now\npresented in the revised Fig. 4A, B and Supplementary Fig. 5A, B, include the requested\nquantitative data with statistical analysis. This rigorous quantification confirms that the\nubiquitination levels of both JAZ1 and JAZ3 proteins in the phyB mutant background\nare indeed significantly more pronounced under the 22°C + JA condition than under\nthe 28°C + JA condition. We have therefore updated the text (Line 280-284) and the\ncorresponding figure legend to accurately reflect these new quantitative findings. We\nbelieve that the statistical analysis now provides clear and objective evidence\nsupporting our conclusions, and we sincerely thank the reviewer for prompting this\nimportant improvement.\n13. Line 283 (Fig. 4D): In phyB mutants, JAZ degradation still occurs in the presence\nof MG132, suggesting an additional proteolytic pathway beyond the proteasome.\nWhile other factors are briefly mentioned in the Discussion, the possibility of\nnon-proteasomal degradation should also be explicitly discussed.\nResponse: We thank the reviewer for this insightful observation. We agree that the\npersistence of JAZ degradation in phyB mutant under MG132 treatment (Fig. 4D)\nsuggested the existence of a compelling, non-proteasomal mechanism. As suggested,\nwe have now explicitly discussed this possibility in the revised Discussion section (Line\n715-719). The added text reads:“Notably, our data demonstrate that the degradation of\nJAZ protein is not fully abolished by MG132. The persistent degradation of the JAZ\nprotein following inhibition with MG132 reveals a layer of complexity in its turnover\nregulation. This key observation strongly argues for the existence of an alternative\ndegradation pathway that operates alongside the ubiquitin-proteasome system. It is\ncrucial to explicitly acknowledge that other mechanisms may contribute to the residual\ndegradation.” Future studies employing specific inhibitors (e.g. E64d for calpains)\nin combination with MG132 will be essential to delineate the relative contributions of\nthese pathways.\n14. Line 297: The statement that “phyB mutant exhibited ~50% lower JAZ levels” is\nnot consistently supported across all data (e.g., Figs. 4F/G appear comparable to WT).\nPresumably, values are normalized to 0 h = 1.0, but this should be stated in the figure\nlegend. Moreover, quantification sometimes appears inconsistent with band intensities.\nPlease re-examine and clarify.\nResponse：We thank the reviewer for this careful observation regarding the of JAZ\nlevel quantifications in the phyB mutant. We have re-performed the grayscale analysis\nand revised the corresponding description in the manuscript (Line 309) and figure\nlegends to clarify that the quantification reflects the relative total protein accumulation\nover time (calculated as AUC) compared to WT. This methodological detail is now\nexplicitly stated, and we believe it reconciles the quantitative conclusion with the\npresented blot images.\n15. Fig. 4A and 4B. Control samples lacking MeJA treatment are missing. Although the\nubiquitin bands may be faint, they should still be shown. Moreover, the rationale for\nusing the inhibitor Bortezomib (Btz) should be explained in the Results, as it likely\ninhibited protein degradation and may have obscured the visualization of the ubiquitin\nbands.\nResponse: We thank the reviewer for the insightful suggestions regarding the\nexperimental design and methodological clarification. In direct response, we have\nrepeated the experiments to include control samples without MeJA treatment, as\npresented in the revised Fig. 4A and 4B and Supplementary Fig. 5A, B. Our updated\ndata clearly show a detectable basal level of JAZ ubiquitination in the absence of\nexogenous MeJA, albeit at a relatively low intensity. Upon MeJA treatment, JAZ\ndegradation is triggered, accompanied by a marked increase in polyubiquitination\nsignal, a pattern consistent with established models of JA-induced degradation.\nThese new results have been incorporated into the revised manuscript (Line 280-284).\nRegarding the use of Bortezomib (Btz), we agree with the reviewer on its critical\nrole in visualizing ubiquitination. As a highly specific and potent proteasome inhibitor,\nBtz blocks the degradation of polyubiquitinated proteins, thereby allowing labile\nubiquitination intermediates—such as those targeting JAZ proteins—to accumulate to\ndetectable levels. This approach is widely adopted to capture transient ubiquitination\nevents. We have now explicitly explained this rationale in the Results section (Line 277-\n280), noting that Btz was selected for its high specificity, efficacy, and reproducibility\nin stabilizing ubiquitinated species.\nWe believe these revisions have significantly strengthened the experimental\nsupport and clarity of our conclusions.\n19\n17. L398–L556. The description of the RNA-seq results is excessively long and should\nbe substantially shortened. The section beginning at line 398 merely lists the number of\ngenes, which is uninformative. The authors must focus on the most essential findings\nand present this section concisely. The section starting at line 508 has the same issue\nand should also be condensed.\nResponse：We thank the reviewer for this constructive feedback regarding the length\nand focus of the RNA-seq results section. We agree that an overly detailed enumeration\nof gene numbers without deeper biological interpretation can diminish the impact and\nclarity of our findings. In response, we have substantially shortened and restructured\nthis section to enhance its conciseness and scientific rigor. The revised context shown\nin the Results section (Line 418-488). We believe these revisions have significantly\nimproved the manuscript’s flow and focus, providing a clearer and more concise\n20\naccount of the critical insights from our transcriptomic analysis.\n18. Fig. 8A, B. Given that a genome-wide analysis of HY5 and MYC2 is already\npresented in Fig. 7, the rationale for performing the Y1H assay is unclear and should\nbe explicitly justified.\nResponse：We thank the reviewer for raising this important point regarding our\nexperimental design. The sequential use of DAP-seq and Y1H assay was intentional, as\neach serves a distinct and critical mechanistic purpose. While DAP-seq is a powerful\ntool for identifying genome-wide potential binding sites in vitro, it cannot definitively\ndistinguish between direct DNA binding and indirect tethering through protein\ncomplexes. The Y1H assay provides essential complementary evidence by\ndemonstrating that HY5 binds the WAG2 promoter directly and independently in a\nminimal yeast system, devoid of plant-specific co-factors or larger protein complexes.\nTherefore, the Y1H assay is not redundant but is crucial for confirming that the WAG2\npromoter contains a bona fide HY5-binding cis-element and to establish direct\ntranscriptional regulation. This layered experimental approach—combining genomic\n(DAP-seq), direct biochemical (Y1H), and in planta phenotypic data—robustly\nestablishes HY5's direct regulatory role.\n19. Fig. 8C–F. The reporter assays were performed only in the wild-type background,\nwhich is insufficient. The authors should also use COI1 and phyB transgenic or mutant\nlines to assess the specific contributions of these factors. Furthermore, it is a significant\nomission that the effects of temperature and MeJA—central to this study—are not\naddressed in these results.\nResponse：Thank you for your valuable comments. We fully agree on the need to\nvalidate the reporter assay results across different genetic backgrounds and to clarify\nthe respective roles of temperature and MeJA. In response, we have performed the\nfollowing key experiments:\n(1) Validation in coi1 and phyB mutant backgrounds: We repeated the reporter assays\nin protoplasts of the coi1-8 and phyB-9 mutants. The results showed that the loss of\nphyB function could composed the transcriptional activation capacity of HY5 but the\n21\nmutation of COI1 did not significantly alter the transcriptional activity of MYC2 on\ntheir downstream targets (such as WAG2, Rap2.6, respectively) compared to the wild-\ntype background. This suggests that the transcriptional activity of HY5 is dependent on\nphyB while MYC2, as key regulatory hubs, may not depend on intact light or JA\nsignaling pathways.\n(2) Analysis of temperature and MeJA effects: We systematically evaluated the\nindividual and combined effects of temperature and MeJA on HY5 and MYC2\ntranscriptional activity in the wild-type protoplast system. The specific findings are as\nfollows: For HY5, its ability of transcriptional activation was not influenced by warm\ntemperature or MeJA treatment. On the contrary, elevated temperature alone (from\n22°C to 28°C) only slightly enhanced the transcriptional activation of MYC2.\nSimilarly, JA treatment alone produced a mild effect of their transcriptional activity.\nCrucially, the strongest synergistic activation effect was observed under the combined\ntreatment of JA and high temperature (28°C). This finding is highly consistent with our\nglobal RNA-seq data and provides direct evidence for elucidating the molecular\nmechanism by which high temperature regulates plant shade avoidance response through\nthe JA signaling pathway.\nThese new experimental data have now been incorporated as Supplementary Fig.\nS18E-H, and the relevant parts of the manuscript have been updated accordingly (Line\n579-592), substantially strengthening the robustness and completeness of our\nconclusions. We thank the reviewer once again for the insightful suggestions, which\nhave significantly enhanced the quality of our work.\n22\n20. Fig. 9. The proposed model is difficult to interpret. To improve clarity, it should be\nredrawn to separately illustrate the scenarios with and without JA under different\ntemperature conditions.\nResponse：We sincerely thank the reviewer for this excellent suggestion. We agree\nthat separating the complex model into distinct scenarios would significantly enhance\nits clarity and interpretability. As suggested, we have now completely redesigned our\nworking model. The new figure set (now Fig. 9) clearly illustrates the molecular\npathways under the four key experimental conditions: Panel A, normal temperature\nwithout JA；Panel B, normal temperature with JA；Panel C, high temperature without\nJA；Panel D, high temperature with JA. This new layout allows for a direct and intuitive\ncomparison of how JA and high temperature individually and interactively regulate the\nkey components of our proposed pathway. We believe this revision has substantially\nimproved the clarity of the proposed model, and we thank the reviewer for the\nconstructive advice.\n1. L54. The term \"Jasmonates\" is generally used.\nResponse: We have made the revision.\n2. L62. Please change \"hormone pathway\" to \"hormone signaling pathway.\"\nResponse: We have made the revision as you suggested.\n3. L78 and L105. Please replace \"Beyond\" with \"In addition to.\"\nResponse: We have made the revision as you suggested.\n4. L124. Please include the relevant citation(s) and elaborate on the biochemical\ncharacteristics and functional significance of 12-HSO₄-JA.\nResponse: We have cited the relevant reference and revised the original text\naccordingly (Line 121).\n5. L145. Please remove the sentence \"JA and high…,\" as this point was already made\nin the Introduction.\nResponse: We have made the revision.\n24\n6. L158. Please change \"selected\" to \"focused on.\"\nResponse: We have made the revision.\n7. L160 and later. Please correct the formatting to remove the space between the number\nand the °C symbol (e.g., 22°C).\nResponse: Thanks for your suggestion. It may be due to a file format conversion issue.\nWe have removed the space between the number and the °C symbol.\n8. Line 166: The phrase “a dramatic reduction in area” appears exaggerated given the\ndata in Fig. 1B. Please moderate the description.\nResponse: We have changed the “dramatic” to “evident”\n9. L168. Please rephrase \"temperature and JA application independently\" to \"either\nelevated temperature or JA application alone.\"\nResponse: We have made the revisions as requested.\n10. L169. Please rephrase \"additive\" to \"additive or synergistic.\"\nResponse: We revised that this is a synergistic effect.\n11. Figures 1C and 1F. Please use ANOVA followed by a two-sided Fisher’s LSD test,\nas comparisons need to be made across all samples.\nResponse: Thank you for your insightful suggestion. As requested, we have re-\nanalyzed the data in Figures 1C and 1F using a two-way ANOVA (factors: JA treatment\nand temperature) followed Tukey's multiple comparison test (p < 0.05) across all\nsample groups. The analysis revealed a statistically significant interaction between JA\ntreatment and high temperature (for Figure 1C: F (1, 8) = 41.78, p = 0.002; for Figure\n1F: F (1, 56) =61.98, p <0.001), indicating that the effect of JA is dependent on the\ntemperature condition and thereby supporting our claim of a synergistic effect.\n12. L261. Please remove \"firstly.\"\nResponse: Thanks. We have removed “firstly”.\n25\n13. L276. Please clarify what is meant by \"either directly or indirectly.\"\nResponse: We have revised the original text (Line 703-705).\n14. Fig. S6E–H. The rationale for these experiments is unclear. Specifically, it should\nbe explained why in vitro experiments were necessary in addition to the in vivo analyses.\nThis point should be described at line 301.\nResponse: We agree that clarifying the necessity of these assays is crucial for readers\nto fully appreciate our experimental design. As noted, in vivo and in vitro approaches\nare complementary. While our in vivo data reveal the physiological state and potential\nfunctions of protein ubiquitination within the native cellular context, the in vitro\nexperiments conducted in a purified system allow us to determine whether the observed\nubiquitination is a direct biochemical outcome, free from potential interference by other\ncellular factors. This combination is essential for establishing a more robust and\nmechanistically clear conclusion.\n15. L441. Please change \"new\" to \"other.\"\nResponse: We have changed “new” to “other”\n16. Throughout the manuscript, the term “JA” is frequently used, but the treatment is\nspecifically MeJA. Please revise for accuracy.\nResponse: The term “JA” was replaced with “MeJA”.\n17. Figure 1E: Treatment conditions (temperature, JA treatment) are not indicated.\nPlease add.\nResponse: We have now revised Figure 1E to explicitly include the treatment labels.\nThis modification ensures that the experimental setup is immediately clear to the reader.\n18. Figures S1 and S2: The number of biological replicates (n) is missing. Please\ninclude this throughout the manuscript.\nResponse：We have added the number of biological replicates.\n26\n19. Figure S3A: Cotyledon area data for 28°C and 22°C + MeJA conditions should be\nadded.\nResponse：We have incorporated the cotyledon area data for 28°C and 22°C+MeJA\nconditions. These new data are now presented in the revised supplementary Fig. 3A.\n20. Figure S4F: The 22°C + JA treatment for 35S:JAZ3-GFP lacks statistical analysis.\nPlease include.\nResponse：Thanks for your advice. We have added the statistical analysis in the revised\nsupplementary Figure 4F.\nReferences\n1. Agrawal R, et al. Mediator subunit 17 integrates jasmonate and auxin signaling pathways\nto regulate thermomorphogenesis. Plant Physiol 189, 2259-2280 (2022).\n2. Huai J, Gao N, Yao Y, Du Y, Guo Q, Lin R. JASMONATE ZIM-domain protein 3 regulates\nphotomorphogenesis and thermomorphogenesis through inhibiting PIF4 in Arabidopsis.\nPlant Physiol 195, 2274–2288 (2024).\nReviewer #1 (Remarks to the Author):\nThis manuscript investigates how temperature cues intersect with jasmonate (JA)\nsignaling to regulate chloroplast-related phenotypes during early seedling development.\nThe authors present an extensive dataset combining genetics, biochemistry, imaging,\ntranscriptomics, and transcription factor binding assays. Compared with the previous\nversion, the revision includes several valuable additions, notably expanded controls and\nin vivo ChIP–qPCR evidence for HY5 and MYC2, which strengthen aspects of the\nproposed regulatory framework.\nHowever, despite these improvements, the manuscript in its current form still suffers\nfrom substantial conceptual, logical, and structural shortcomings. These issues do not\nnecessarily require major new experiments, but they do require significant tightening\nof mechanistic precision, narrative logic, and interpretative restraint, as well as\nsubstantial condensation of the text.\nMajor issues：\n1. The current title, “Phytochrome B and CORONATINE INSENSITIVE1 integrate\nhigh ambient temperature and jasmonate signaling to regulate chloroplast\ndevelopment”, the title positions phyB and COI1 as equivalent “integrators” of\ntemperature and JA signaling. However, the data support an integrative role only for\nphyB, a known thermoresponsive photoreceptor. COI1 functions as a canonical JA\nreceptor and is not shown to sense temperature or to act as a signal integration node.\nThis framing is inconsistent with the revised manuscript, particularly given that claims\nof direct phyB–COI1 interaction were removed. The data demonstrate temperature-\ndependent modulation or gating of JA signaling output via phyB, rather than\nsymmetric or equivalent integration of temperature and hormone signals at multiple nodes.\nResponse: We thank this reviewer’s good comment on the manuscript title and have\nrevised it to “Phytochrome B integrates jasmonic acid and warm temperature signaling\npathways to regulate cotyledon chloroplast development”.\n2. The Introduction (line 76-79) currently states that phyB converts from its biologically\nactive Pfr form to its inactive Pr form “upon exposure to high ambient temperature.”\nThis wording implies a temperature-triggered, switch-like Pfr → Pr conversion,\nanalogous to light-induced photoconversion, and is therefore mechanistically\ninaccurate. Current evidence indicates that phyB temperature responsiveness arises\nfrom temperature-dependent acceleration of Pfr dark reversion, resulting in shifts in the\nPfr/Pr equilibrium and nuclear phyB activity under warm conditions, rather than from\na discrete temperature-triggered conversion event. Moreover, recent molecular and\nbiophysical work (e.g., Zhong et al., Molecular Cell, 2022) emphasizes that phyB\nthermosensing involves modulation of phyB kinetic and physical properties (including\ncondensate behavior), reinforcing the view that phyB temperature sensing is governed\nby thermodynamic and kinetic regulation rather than an active thermal switch.\nBesides, other parts of the Introduction should also be carefully revised accordingly to\nreflect this more accurate mechanistic understanding.\nResponse: We are grateful for the reviewer pointing out the incorrect claim of thermo-\nregulation of phyB. We have cited addition references and revised the statement as\n“phyB also exhibits light-independent Pfr/Pr dynamic thermal reversion and the relative\namount of the Pr form is accelerated with increasing temperature (Klose et al., 2020).\nA recent study reveals that, while light induces phyB photobody formation, the N-\nterminal extension of phyB acts as a biophysical modulator of phase separation to\ndirectly sense warm temperature signal (Chen et al., 2022). This temperature\ndependence of thermal reversion provides the basis for phyB to act as thermosensor\ncontrolling plant elongation growth (Jung et al., 2016; Legris et al., 2016)”.\nMoreover, we have re-organized the Introduction part in the order of phyB-mediated\nwarm temperature signaling, the JA signaling pathway, and their crosstalk. We believe\nthat this revision of the Introduction reflects more accurate mechanistic understanding\nto the readers.\n3. The working model presented in Fig. 9, together with its accompanying legend,\ncontains multiple interrelated mechanistic inconsistencies that substantially undermine\nthe credibility of the proposed pathway and should be addressed in a unified manner.\nFirst, the model depicts warm temperature as inducing a discrete transition of phyB into\nan “inactive” state. This representation is mechanistically imprecise and potentially\nmisleading. Warm temperature does not trigger a photoconversion-like or switch-like\ninactivation of phyB. Instead, current evidence indicates that phyB temperature\nresponsiveness arises from temperature-dependent acceleration of Pfr dark reversion,\nresulting in a shifted Pfr/Pr equilibrium (Legris et al., Science 2016) and reduced\nsteady-state nuclear phyB activity under warm conditions (Zhong et al., Molecular Cell,\n2022). Portraying phyB as simply “inactive” therefore reinforces an incorrect switch-\nbased interpretation rather than a kinetic and thermodynamic one. Notably, this same\noversimplification is mirrored in the Introduction, where phyB is described as\nconverting from Pfr to Pr “upon exposure to high ambient temperature,” indicating a\nbroader conceptual issue that affects both text and model.\nSecond, the model implicitly assumes that HY5 protein levels remain stable under\nwarm temperature, with temperature effects being mediated primarily through MYC2\nactivation. This assumption is not supported and is inconsistent with published evidence\ndemonstrating that high ambient temperature promotes HY5 protein degradation\n(Frontiers in Plant Science, 2017). HY5 is therefore itself a temperature-sensitive node\nat the protein level, rather than a static component whose function is merely overridden\nby MYC2. As currently depicted, the model oversimplifies temperature signaling by\nselectively assigning temperature responsiveness to MYC2 while leaving HY5\nunaffected.\nThird, and most critically, the phyB–JAZ–MYC2 relationship described in Fig. 9 and\nits legend contains a fundamental logical error. The legend states that under JA\ntreatment at ambient temperature, “active phyB stabilizes JAZ repressors, which\nreduces their inhibition of MYC2.” This statement is mechanistically incorrect on two\nlevels. Under canonical JA signaling, the presence of JA leads to COI1-dependent\nubiquitination and degradation of JAZ proteins, rather than their stabilization,\nirrespective of phyB status. Moreover, even if JAZ proteins were stabilized, this would\nbe expected to enhance, not reduce, repression of MYC2. Thus, the causal logic\npresented in the model is internally contradictory and reverses the established\ndirectionality of JAZ–MYC2 regulation.\nWhat’s more, throughout the manuscript and figures (notably Fig. 9), 22°C is\nrepeatedly referred to as “ambient temperature.” In the context of controlled growth\nexperiments, this terminology is misleading, as “ambient” typically implies fluctuating\nor natural environmental conditions. The authors should standardize terminology to\n“control/standard temperature (22°C)” and “warm or high ambient temperature (28°C)”,\nprovide a clear definition upon first use, and apply this terminology consistently across\nthe main text, figures, legends, and Supplementary Information.\nTaken together, Fig. 9 presents temperature–JA crosstalk as a largely linear, state-\nswitch–driven pathway, whereas available evidence supports a more complex\nregulatory scenario involving multiple temperature-sensitive nodes, kinetic modulation,\nand shifting transcription factor balance. The current model therefore overstates\nmechanistic certainty and, in several places, directly conflicts with well-established\nsignaling logic. The authors should substantially revise Fig. 9 and its legend to (i) avoid\ndepicting phyB temperature sensing as a discrete inactive state, (ii) explicitly reflect\ntemperature-dependent kinetic regulation of phyB, (iii) account for or appropriately\nqualify the temperature sensitivity of HY5, and (iv) correct the phyB–JAZ–MYC2\nrelationships to ensure consistency with canonical JA signaling. The revised model\nshould be clearly presented as a working hypothesis, rather than as a resolved\nmechanistic pathway.\nResponse: We sincerely thank this reviewer for providing very valuable suggestions on\nthe working model. According to these comments and based on our results, we have\nrevised the working model in Fig. 9. We streamlined the experimental design by\nfocusing on two representative conditions: normal growth and combined treatment\n(28℃+MeJA), which allowed us to more directly elucidate the relationship between\nphyB and JAZ proteins, as well as the distinct regulatory roles of MYC2 and HY5 on\ndownstream gene expression. Through this refined model, we are able to clearly\ndemonstrate how plants dynamically balance growth and defense via integrated JA and\nwarm temperature signaling, revealing a coherent adaptive strategy in response to\nenvironmental pressures.\nIn addition, we provide a clear definition on temperature conditions upon first use,\nnormal growth temperature (22°C) and warm temperature (28°C), and apply this\nterminology consistently across the full text.\n4. While individual experiments in the Results section are generally well executed, the\noverall narrative flow is difficult to follow due to the absence of explicit logical\ntransitions between subsections. Experimental blocks often appear juxtaposed rather\nthan sequentially motivated, leaving it unclear how each new set of experiments arises\nfrom the preceding findings.The authors should add concise transition sentences or\nshort paragraphs that explicitly state the biological question motivating each subsequent\nexperimental block, thereby clarifying the progression from phenotypic observations to\nmechanistic conclusions.\nResponse: We thank these nice suggestions and have revised the Result sections by\nadding short parapraphs or concise transition sentences, at the beginning of each\nsubsections, that explicitly explain the question and motivation to the subsequent\nexperiments and studies. For examples, “These photosynthetic fluorescence changes\nprompted us to further determine the effect on chloroplast development”; “We\nhypothesized that phyB might have functional relationship with JAZs. To test this, …”.\nThese revisions help clarifying the progression in a logical manner.\n5. Despite claims of global adoption of two-way ANOVA for factorial designs, several\nSupplementary figures still rely on Student’s t-tests in contexts involving multiple\nvariables or time courses. This inconsistency should be resolved either by appropriate\nreanalysis or by explicit justification.\nResponse: We have reanalyzed the related statistical analysis according to this comment.\n6. The main text (~8,000 words, excluding Methods and figure legends) is excessively\nlong for a primary research article and is difficult to read in a single pass. This length\nis driven primarily by redundant description, diffuse framing, and repetition between\nthe Results and Discussion, rather than by exceptional conceptual complexity. The\nauthors should substantially condense and streamline the manuscript, particularly the\nResults and Discussion, by removing redundant descriptions, tightening interpretations,\nand clearly prioritizing core conclusions over secondary observations. Streamlining\nwould strengthen, rather than weaken, the manuscript.\nResponse: We are grateful for these suggestions and agree with this reviewer that the\nlength of our previous manuscript is too long. According to these suggestions, we have\ncarefully and extensively condensed and streamlined the Results and Discussion, by\nremoving redundant descriptions, tightening interpretations, and prioritizing core\nconclusions. Thus the manuscript length has been significantly shortened. We believe\nthat these revisions greatly strengthen and improve the readability of the manuscript\nwithout weakening or lose the conclusions of this story.\n7. In addition to the points listed above, the manuscript contains numerous minor issues\nrelated to grammar, wording, spacing, and formatting throughout the text. While none\nof these issues individually undermine the scientific conclusions, their cumulative\neffect substantially affects readability and gives the impression that the manuscript has\nnot been carefully proofread. I do not list these issues exhaustively here, but strongly\nencourage the authors to carefully reread the entire manuscript, ideally with thorough\nlanguage editing, to correct grammatical errors, inconsistent spacing, and other\ntypographical problems prior to resubmission.\nResponse: This is a nice comment. We have made careful proofreading and formatting\nthroughout the entire manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors have made a substantial effort to address the concerns raised in the previous\nreview. A large number of additional experiments have been performed, including new\nlight-quality analyses, expanded genetic controls, additional biochemical assays, and a\nrestructured working model. Overall, the revised manuscript is significantly improved\nin terms of mechanistic depth, data presentation, and clarity. The integration of the\nphyB–JAZ module with downstream HY5/MYC2 signaling is now more convincing\nthan in the original submission. Nevertheless, a few issues still require attention before\nthe manuscript can be considered fully satisfactory.\nIn lines 564–567, the extensive listing of plasmid constructs interrupts the narrative\nflow and reduces readability. Condensing this information or relocating it to the\nMethods or a supplementary table would improve clarity.\nResponse: We thank this comment and delect the construct information from the Results\nsection to condense the experiment description and to improve readability.\nIn Figure 9, the working model would benefit from a clearer visual distinction between\ninteracting and non-interacting molecular components, for example by depicting\ninteracting molecules in close physical proximity and separating those that do not\ninteract.\nResponse: The working model in Figure 9 has been revised as the two reviewers\nsuggested.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\n and to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nReviewer #1 (Remarks to the Author):\nI have carefully read the revised version of the manuscript. Overall, the authors have\nmade substantial efforts to address the major conceptual and mechanistic concerns\nraised in previous rounds, and the core conclusions are now generally supported by the\npresented data. I do not have further major scientific objections, and I consider the study\nsuitable for publication after minor editorial refinement. This version of the manuscript\nwould still benefit from careful language polishing and stylistic tightening, as there\nremain numerous minor issues that affect readability. These issues do not undermine\nthe conclusions, but addressing them would significantly improve the clarity and\nprofessionalism of the final paper.\nThroughout the manuscript, there are sentences that are overly long or syntactically\ncomplex, making them difficult to follow. In several places, clauses are stacked without\nclear logical connectors, and some sentences would benefit from being split into shorter,\nmore direct statements.\nCertain terms are used repeatedly in a vague or redundant manner (e.g., “suggest,”\n“indicate,” “play an important role,” “is involved in”), sometimes within the same\nparagraph. Tightening wording and reducing repetition would improve precision and\nreadability.\nWhile temperature-related terminology has improved compared with earlier\nversions, minor inconsistencies in phrasing remain across sections (main text vs. figure\nlegends vs. Supplementary Information). A final consistency check is recommended.\nThe overall narrative flow has improved, but some paragraph transitions, particularly\nin the Results and Discussion, remain abrupt. Adding brief linking phrases would help\nguide readers through the logic more smoothly.\nResponse: We are grateful for the valuable comments provided by this reviewer. In this\nrevised manuscript, we have thoroughly and carefully polished the writing, split some\nlong sentences, tightened relatived wording to minimize redundancy. The temperature-\nrelated terminology has been clearly defined across sections, revised as warm\ntemperature."}]

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