Transcriptomic Profiling of Heat-Treated Oriental Lily Reveals LhERF109 as a Positive Regulator of Anthocyanin Accumulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper presents innovative information on how anthocyanin synthesis genes transcription responds to heat stress in oriental lily. The results can be best explored and figures legends needs to be revised. More details on the methods section should be given to allow reproducibility. Some conclusions are too speculative, and should be rewritten. Specific comments are listed below.
-------
The supplementary material is never cited in-text. This should be corrected.

Introduction
Lines 57-58: Reference 9 refers to red pear and not Malus domestica, as stated in the text. Please, revise this citation.
Lines 90-96: the papers should be cited here to corroborate these statements

Methods
Line 104: what do these acronyms mean? More details on the developmental stages should be given to improve clarity to the reader. I suggest Fig. 1A to be cited here
What were the growth conditions in which these plants were maintained prior to treatments? This should be clarified to allow reproducibility
Line 107: What is the level of photosynthetic flux density? Please, replace lux values for PFD.  The light intensity in lux measurements varies according to the type of lamps, so it may hamper reproducibility.
Line 108: more details on sampling are required to allow reproducibility - how many tepals, from how many individuals, were sampled per biological replicate? Were the samples frozen in liquid nitrogen during sampling, followed by storage in a deep freezer?
Line 118: what is meant by ‘relative units’?
Line 187: more details on Agrobacterium inoculum preparation are required to allow reproducibility; perhaps cite the methods from a previous paper or include the details as supplementary material.

Results
Line 210-211: Fig. 1B should be cited at the end of the first sentence. To improve fluidity in the text and to facilitate the understanding for the reader, include the time points e.g. “In sharp contrast, upon initiating the 35℃ treatment, the red coloration of the lily petal began to decline from 12h of stress” 
Legend for figure 1 explain what is T1-T4. The description of sub-figure C and D are incorrect regarding the figure, and there is no mention of item E. Please, revise.
Lines 217-220: No mention of the reducing and soluble sugars results is included here.
Line 221: Since qPCR data are shown before the RNA-seq, a more detailed description of the results should be given here. Then, after the RNA-seq data, the information that the pattern in transcriptomic analyses is corroborated by qPCR should be described. 
Figure 3: the legend states that T1-T4 refers to different time points from those on the methods section. Please, revise.
Some wrong wording was found, e.g. ‘lilies’ and not ‘lillies’
Line 291: the mention of LhERF109 here feels out of place, since this gene is not reference at all in this section of GO analysis; I suggest this sentence is removed
Figure 5 legend: it says apple and not lilly; please, revise. In Fig. 5A what do the colors in the x axis mean? In Fig. 5B, what does Cy and TCA mean? More information on the legend should be given.
In the description of figure 5, it should be highlighted where LhERF109 is placed among all the data and how this gene relates to the other genes. I believe it is too vague as it is.
Line 344: there is no figure 6A – revise.
The phylogenetic tree is not well explored. There is only one sentence describing its results. I believe it should be moved to supplementary results.

Discussion
This section is too descriptive. This should be reduced to prevent repetition with the results section – e.g. paragraph in line 424.
Line 413: this interpretation is too speculative, as isoflavone synthesis was not addressed in the results. If this is to be discussed, more focus on isoflavone synthesis genes should be given in the RNA-seq results section.
Line 435 : Figures should not be cited in the discussion section.
Line 478: this is speculative. For this to be confirmed, knockout or OE lines on the LhERF109 gene should be designed. 

Author Response

Author's response to the reviewer 1

Dear Reviewer 1, 

We greatly appreciate your thorough and constructive review of our manuscript “Transcriptomic Profiling of Heat-Treated Oriental Lily Reveals LhERF109 as a Positive Regulator in Anthocyanin Accumulation” submitted to Agronomy. Your insightful comments have been invaluable in improving the clarity, rigor, and overall quality of our work.

To facilitate your review, we have highlighted all revisions in the revised manuscript using red font. Below, we provide detailed point-by-point responses to each of your comments:

Introduction  

Comments 1:

Lines 57-58: Reference 9 refers to red pear and not Malus domestica, as stated in the text. Plase, revise this citation.

Response:

Thank you for identifying this citation error. You are correct that Reference 9 (Bai et al., 2019) studied anthocyanin regulation in red pear (Pyrus pyrifolia), not apple (Malus domestica) as we incorrectly stated in our manuscript. We have corrected this error and properly revised the citation. The revised text now reads (lines 57-63): "In apple (Malus domestica), the interaction between high temperature and light signaling involves B-box (BBX) transcription factors, with MdCOL11 (BBX22 homolog) directly promoting MdMYB10 transcription under light conditions while being suppressed by high temperatures [9], whereas MdCOL4 (BBX24 homolog) is induced by heat shock transcription factors (HSF) under high temperatures and forms a MdCOL4-MdHY5 dimer that inhibits the expression of ANS, UFGT, and MdMYB1/MdMYB10 genes [10]."

9. Bai, S.; Saito, T.; Honda, C.; Hatsuyama, Y.; Ito, A.; Moriguchi, T. An apple B-box protein, MdCOL11, is involved in UV-B- and temperature-induced anthocyanin biosynthesis. Planta 2014, 240, 1051–1062.
10. Fang, H.; Dong, Y.; Yue, X.; Chen, X.; He, N.; Hu, J.; Jiang, S.; Xu, H.; Wang, Y.; Su, M.; Zhang, J.; Zhang, Z.; Wang, N.; Chen, X. MdCOL4 interaction mediates crosstalk between UV-B and high temperature to control fruit coloration in apple. Plant Cell Physiol. 2019, 60, 1055–1066.

Comments 2:

Lines 90-96: the papers should be cited here to corroborate these statements

Response:

Thank you for this suggestion. We have added appropriate citations to support our statements about flower color fading in Oriental lilies. The revised text now reads (lines 89-95): "However, pink-flowered cultivars commonly exhibit heat sensitivity, with flower color fading under high temperature stress [28]. This undesirable trait is often inherited by progeny, significantly reducing product quality and economic value. While research has established that MYB transcription factors, particularly LhMYB12 and LhMYBC2 [4,28], play key roles in regulating flower color fading in Oriental lilies under high temperature, the upstream molecular networks controlling these MYB factors remain largely unexplored."

References [4] and [28] specifically support our statements about the role of MYB transcription factors:

 

4. Yang, J.; Guo, C.; Chen, F.; Lv, B.; Song, J.; Ning, G.; He, Y.; Lin, J.; He, H.; Yang, Y.; Xiang, F. Heat-induced modulation of flavonoid biosynthesis via a LhMYBC2-Mediated regulatory network in oriental hybrid lily. Plant Physiol. Biochem. 2024, 214, 108966.
5. Lai, Y-S.; Yamagishi, M.; Suzuki, T. Elevated temperature inhibits anthocyanin biosynthesis in the tepals of an Oriental hybrid lily via the suppression of LhMYB12 transcription. Sci. Hortic. 2011, 132, 59–65.

We have maintained the statement regarding the inheritance of heat sensitivity without a specific citation as this represents common horticultural knowledge observed in breeding programs across various Oriental lily hybrids and their progeny (such as LLO and OOT cultivars). This observation is widely accepted among lily breeders and growers, though specific studies quantifying this inheritance pattern are limited in the literature.

Methods  

Comments 3:

Line 104: what do these acronyms mean? More details on the developmental stages should be given to improve clarity to the reader. I suggest Fig. 1A to be cited here. What were the growth conditions in which these plants were maintained prior to treatments? This should be clarified to allow reproducibility.

Response:

Thank you for your insightful comments. In response, we have revised the manuscript to clarify the developmental stages, define acronyms, and provide detailed plant growth conditions to enhance reproducibility. The revised text now reads (lines 103-113).

We have now defined the floral developmental stages as follows: St2 represents the early tepal elongation stage with slight pigmentation, and St3 corresponds to further tepal expansion accompanied by increasing tepal coloration. This information has been added to the Methods section, and a citation to Fig. 1A has been included for visual reference (lines 106-108).

Additionally, we have added a detailed description of the pre-treatment growth conditions. Specifically, Lilium 'Souvenir' plants were cultivated under standard greenhouse conditions (20 ± 2°C, 65 ± 5% relative humidity, natural photoperiod supplemented with artificial lighting to maintain a minimum of 200 μmol m⁻² s⁻¹ PPFD for 16 hours daily) from bulb sprouting until the designated developmental stages were reached. These clarified details have been incorporated into the revised manuscript at lines 103-106.

We also specified the environmental parameters used in the growth chambers during temperature treatments (20°C vs. 35°C, 16h light/8h dark photoperiod, 70% relative humidity, 200 μmol m⁻² s⁻¹ PPFD) and clearly described the sampling protocol, including biological replication and storage procedures. This ensures the experiment can be reliably reproduced (lines 111).

Comments 4:

Line 108: more details on sampling are required to allow reproducibility - how many tepals, from how many individuals, were sampled per biological replicate? Were the samples frozen in liquid nitrogen during sampling, followed by storage in a deep freezer?

Response:

We have added detailed information about our sampling procedure in the revised manuscript. The revised text now reads (lines 112-116): "For each biological replicate, three inner tepals were collected from each of three individual flowers (nine inner tepals total per biological replicate), with three biological replicates per treatment combination. Immediately after collection, samples were flash-frozen in liquid nitrogen and stored at -80°C until further physiological and molecular analyses were performed."

We specifically chose to sample inner tepals rather than outer tepals because inner tepals are protected from direct light exposure during early flower development, resulting in more uniform initial pigmentation. Outer tepals, which are exposed to light during bud development, often show pre-existing pigmentation patterns and physiological adaptations to light stress that could potentially mask or interfere with the temperature-specific responses we aimed to study. Previous studies in lilies have also demonstrated that inner tepals exhibit more consistent and pronounced responses to temperature treatments (Yamagishi, 2022). This sampling strategy allowed us to more accurately capture the direct effects of temperature on tepal pigmentation development with minimal confounding variables

References: Yamagishi, M. (2022). High temperature enhances anthocyanin coloration in Asiatic hybrid lily flowers via upregulation of the MYB12 positive regulator. Horticultural Plant Journal, 8(3), 281-289.

Comments 5: 

Line 118: what is meant by 'relative units'?

Response:

Thank you for this question. We have clarified the units of anthocyanin measurement in our revised manuscript. In line 124, the term "relative units" has been replaced with "absorbance units per gram fresh weight" to precisely indicate that our measurements represent spectrophotometric values normalized to tissue weight, rather than absolute concentration values determined through HPLC with authentic standards. This terminology better reflects the nature of our quantification method.

Comments 6: 

More details on Agrobacterium inoculum preparation are required to allow reproducibility; perhaps cite the methods from a previous paper or include the details as supplementary material. 

Response:

We thank the reviewer for this helpful comment. To improve clarity and reproducibility, we have substantially revised Section 2.7 to provide detailed information on the Agrobacterium tumefaciens GV3101 inoculum preparation and infiltration procedures. Specifically, we have added the antibiotic concentrations used for bacterial culture (rifampicin and kanamycin), OD₆₀₀ values at both growth and infiltration stages, the complete composition of the infiltration buffer (10 mM MES, 10 mM MgCl₂, 150 µM acetosyringone, pH 5.6), and the incubation conditions (room temperature, 3 h, dark) required to induce virulence gene expression. We also clarified the specific mixing protocol for pTRV1 and pTRV2/pTRV2-LhERF109 used in VIGS experiments (line 197-207).

In addition, we refined the description of the infiltration process and post-treatment handling, including the use of developmental stage St2 flower buds, infiltration volume, injection method, replicate number, hydration conditions, growth chamber settings (temperature, photoperiod, humidity), and the timing of phenotypic and gene expression assessments. These additions have been made to ensure that the experimental procedures are fully transparent and reproducible by other researchers (line 208-222).

References:

47. Yin, X.; Zhang, Y.; Zhang, L.; Wang, B.; Zhao, Y.; Irfan, M.; Chen, L.; Feng, Y. Regulation of MYB Transcription Factors of Anthocyanin Synthesis in Lily Flowers. Front. Plant Sci. 2021, 12, 699303.

And we would like to clarify that while the transcriptome analysis was performed using the Oriental lily cultivar ‘Souvenir’, the transient transformation and VIGS experiments were carried out using the cultivar ‘Sorbonne’. This substitution was necessary due to the declining commercial availability of ‘Souvenir’, which has become increasingly difficult to obtain.Importantly, both ‘Souvenir’ and ‘Sorbonne’ are pink-flowered Oriental lily cultivars that exhibit similar floral pigmentation patterns and anthocyanin fading in response to high temperature. Our previous observations and comparative transcriptomic analyses support the conclusion that heat-induced flower color fading is not a phenomenon restricted to a single cultivar, but rather a shared physiological response across pink-flowered Oriental lilies. Therefore, the regulatory mechanisms—particularly those involving LhERF109—are likely conserved within this group. We have added a brief clarification in the Materials and Methods section (Line 207-211) .

Results

Comments 7: 

Line 210-211: Fig. 1B should be cited at the end of the first sentence. To improve fluidity in the text and to facilitate the understanding for the reader, include the time points e.g. "In sharp contrast, upon initiating the 35℃ treatment, the red coloration of the lily petal began to decline from 12h of stress" 

Response:

Thank you for this helpful suggestion to improve the clarity and flow of our manuscript. We have revised the text according to your recommendation by moving the Fig. 1B citation to the appropriate position and incorporating the specific time point information. The updated text now reads (lines 240-243):

"Under the 20℃ treatment, the St2 of the lily 'Souvenir' exhibited a progressive petal coloration phenomenon (Figure 1A). In sharp contrast, upon exposure to the 35℃ treatment, the red pigmentation of the lily petals began to decline after 12h of heat stress, and subsequently continued to fade, eventually approaching an almost white appearance (Figure 1B)."

This revision improves the temporal context of our observations and enhances the readability of this section for readers.

Comments 8:

Legend for figure 1 explain what is T1-T4. The description of sub-figure C and D are incorrect regarding the figure, and there is no mention of item E. Please, revise.

Response:

Thank you for pointing out these inconsistencies in our Figure 1 legend. We have thoroughly revised the legend to properly explain T1-T4 time points and to accurately describe all sub-figures including panel E, which was previously omitted. The revised legend now reads(lines 265-27):

"Figure 1. Phenotypes, pigment, sugar content, and gene expression changes in lily ‘Souvenir’ petals under control and high-temperature treatments. (A) Diagram of the developmental stages of lily 'Souvenir' (St1: bud tight stage, St2: initial coloration stage, St3: partial coloration stage, St4: full coloration stage, 0 d: 0 day of flowering, 1 d: 1 day of flowering) Scale bar = 5 cm. (B) Comparison of lily 'Souvenir' petal color under control (CK, 20℃) and high-temperature (HT, 35℃) treatments at different time points (T1: 12h, T2: 24h, T3: 48h, and T4: 72h) Scale bar = 5 cm.  (C) Total anthocyanin content and Cyanidin 3-O-β-rutinoside content in lily 'Souvenir' petals under control (CK, 20℃, blue) and high-temperature (HT, 35℃, red) treatments at time points T1-T4. (D) Sugar content in lily 'Souvenir' petals under control (CK, 20℃, blue) and high-temperature (HT, 35℃, red) treatments at time points T1-T4, including soluble sugar content (left), reducing sugar content (middle), and sucrose content (right). (E) Relative expression levels of structural genes and transcription factors from transcriptome analysis validated by qPCR in lily 'Souvenir' petals under control (CK, 20℃, blue) and high-temperature (HT, 35℃, red) treatments at time points T1-T4 (* p < 0.1, ** p < 0.01, *** p < 0.001)."

Comments 9:

Lines 217-220: No mention of the reducing and soluble sugars results is included here.

Response:

Thank you for pointing out this omission in our manuscript. We have revised the text to include information about all sugar measurements shown in Figure 1D. The updated text now acknowledges the results for soluble sugar and reducing sugar contents while emphasizing their limited relationship with petal color changes compared to sucrose.The reason for this approach is that previous studies have also indicated that soluble sugars and reducing sugars do not show a significant correlation with flower color changes under high temperature conditions. While we now acknowledge these measurements as they appear in Figure 1D (addressing your concern about their omission), we do not elaborate further on these results since they are less relevant to the color change mechanism being investigated. Our focus remains on sucrose content, which demonstrates a more meaningful relationship with the observed phenotypic changes in petal coloration under temperature stress.

The revised text now reads (lines 247-252): "The determination of the changes in the sugar content of Oriental lily 'Souvenir' flowers under high temperature showed that over time, the sucrose content in the petals generally increased under suitable temperature conditions (20°C), while it generally decreased under high temperature conditions (35°C). Although soluble sugar and reducing sugar contents also varied between treatments, their relationship with petal color changes was not as pronounced as that of sucrose (Figure 1D)."

Comments 10:

Line 221: Since qPCR data are shown before the RNA-seq, a more detailed description of the results should be given here. Then, after the RNA-seq data, the information that the pattern in transcriptomic analyses is corroborated by qPCR should be described.

Response:

Thank you for this suggestion. We have revised the text to provide a more detailed description of the qPCR results before discussing how they align with the RNA-seq data. The revised text now reads (lines 253-263): 

"To examine the expression patterns of genes involved in anthocyanin biosynthesis under different temperature conditions, this study conducted qPCR analysis of key structural genes and transcription factors. The qPCR results (Figure 1E) demonstrated that high temperature (35 ℃) treatment led to a dramatic downregulation of all six analyzed genes compared to control conditions (20 ℃). The expression levels of structural genes UGT2, CHS, DFR, F3H, and ANS were severely suppressed under high temperature across all time points (T1-T4), with expression reduced to nearly undetectable levels. Similarly, the transcription factors MYB12 and bHLH2 also exhibited significant downregulation under high temperature stress. The subsequent transcriptomic analysis corroborated these qPCR findings, indicating that the sequencing results obtained in this study exhibit a high level of reliability."

This revision provides a comprehensive description of the qPCR results shown in Figure 1E before mentioning their correlation with the RNA-seq data that appears later in the manuscript.

Comments 11:

Figure 3: the legend states that T1-T4 refers to different time points from those on the methods section. Please, revise.

Response:

Thank you for your careful review and for pointing out this inconsistency. We apologize for the confusion regarding the time points in Figure 3. You are correct that there is a discrepancy between the time points described in the figure legend and those in the methods section. We have revised the Figure 3 legend to ensure consistency with the methods section and other figures throughout the manuscript. The corrected legend now reads(lines 342):

"Figure 3. Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) in Oriental lily tepals under high temperature stress. The DEGs were categorized into three main types: biological process (BP), cellular component (CC), and molecular function (MF). T1, T2, T3, and T4 represent samples collected at 12h, 24h, 48h, and 72h after 20°C and 35°C treatment, respectively. The color scale indicates -log10(p-value) of enriched GO terms, and the size of dots represents the count of genes in each term."This revision ensures that the time points (T1: 12h, T2: 24h, T3: 48h, and T4: 72h) are consistent throughout all figures and sections of the manuscript."

This revision ensures that the time points (T1: 12h, T2: 24h, T3: 48h, and T4: 72h) are consistent throughout all figures and sections of the manuscript.

Comments 12:

Some wrong wording was found, e.g. ‘lilies’ and not ‘lillies’

Response:

Thank you for pointing out the potential spelling error regarding "lilies" versus "lillies". After thoroughly reviewing the entire manuscript, we have not been able to locate any instances where "lillies" (with double 'l') appears in our text. We have confirmed that all occurrences of this term are correctly spelled as "lilies" with a single 'l'. To ensure we address this concern properly, could you please specify the exact line number or section where you observed this spelling error? This would help us make the necessary correction with precision.

Comments 13:

Line 291: the mention of LhERF109 here feels out of place, since this gene is not reference at all in this section of GO analysis; I suggest this sentence is removed

Response:

Thank you for this observation. We agree that the mention of LhERF109 is incongruous with the GO analysis section, as this gene is not referenced elsewhere in this context. We have removed this sentence from the manuscript as suggested, which improves the coherence and focus of the GO analysis section.

Comments 14:

Figure 5 legend: it says apple and not lilly; please, revise. In Fig. 5A what do the colors in the x axis mean? In Fig. 5B, what does Cy and TCA mean? More information on the legend should be given.

Response:

We sincerely thank the reviewer for pointing out the issues in the Figure 5 legend. The revised text now reads (lines 386-398):

(1) In Figure 5A, the colors on the x-axis below the dendrogram represent different co-expression modules identified by WGCNA. Each color corresponds to a unique gene module, and genes within the same module share similar expression patterns.

(2) In Figure 5B, we have clarified the trait abbreviations: "C3G" refers to cyanidin-3-O-glucoside content and "TAC" refers to total anthocyanin content. These definitions have now been explicitly added to the revised figure legend.

We have revised the entire Figure 5 legend to include these clarifications. The updated legend now provides a clearer and more comprehensive explanation of each panel, including correlation thresholds, visual encodings, and specific gene annotations such as LhERF109.

Comments 15:

Line 344: there is no figure 6A – revise.

Response:

Thank you for pointing out this inconsistency. You are correct that there is a labeling error in line 344 where we reference "Figure 6A" which does not exist with this specific label. We have corrected this reference to simply "Figure 6" to accurately reflect our figure numbering system. This correction ensures proper citation of our results and improves the clarity of our manuscript (Line 403).

Comments 16:

The phylogenetic tree is not well explored. There is only one sentence describing its results. I believe it should be moved to supplementary results.

Response:

Thank you for your comment regarding the phylogenetic tree. While we understand your concern about the limited description, we believe that maintaining this analysis in the main text is crucial rather than moving it to supplementary results. The phylogenetic positioning of LhERF109 within ERF subfamily Group X and its close relationship with AtERF109 (with 92% bootstrap support) provides essential evolutionary context for our functional studies. This relationship directly informs our mechanistic investigations and is referenced in our discussion of LhERF109's role in stress response pathways. We have expanded the description to better highlight these important evolutionary relationships and their relevance to our functional findings. This phylogenetic evidence serves as a foundational element that connects our work to the established literature on ERF transcription factors and their conserved functions across plant species.

Discussion 

Comments 17:

This section is too descriptive. This should be reduced to prevent repetition with the results section – e.g. paragraph in line 424.

Response:

Thank you for this valuable suggestion. We agree that the original paragraph contained redundant descriptions already presented in the Results section. To address this, we have revised the paragraph (now located in lines 491–497 of the Discussion) to focus on the evolutionary implication of LhERF109, particularly its homology with AtERF109 in Arabidopsis thaliana and its potential role in hormone signaling. This revised version removes descriptive elements about gene expression and emphasizes broader functional relevance, thereby avoiding unnecessary repetition. The corrected legend now reads:

“While our findings are rooted in Oriental lily, the regulatory model parallels broader insights from other plant systems. Notably, LhERF109 shares high sequence similarity with AtERF109, a Group X ERF transcription factor in Arabidopsis thaliana known to mediate jasmonic acid and auxin crosstalk and to function in stress-responsive developmental processes [59]. This homology suggests that LhERF109 may act as a lily-specific analog, integrating hormone signaling pathways into the transcriptional regulation of anthocyanin biosynthesis.”

 

Comments 18:

Line 413: this interpretation is too speculative, as isoflavone synthesis was not addressed in the results. If this is to be discussed, more focus on isoflavone synthesis genes should be given in the RNA-seq results section.

Response:

Thank you for your critical comment. We acknowledge that our earlier statement on isoflavone synthesis lacked sufficient experimental support. In the revised version, we have removed direct assumptions regarding isoflavone metabolism and instead reframed the paragraph to emphasize that our transcriptomic data indicate a broader transcriptional suppression of flavonoid metabolism under heat stress, without making specific claims about isoflavone synthesis. This revision avoids overinterpretation while maintaining alignment with the main findings of our study.

The revised paragraph now appears in lines 470–478 of the Discussion section.

Comments 19:

Line 435 : Figures should not be cited in the discussion section.

Response:

Thank you for pointing out the issue with figure citations in the discussion section. We have addressed this by removing all figure references from our discussion, specifically removing the figure citations that appeared in discussion. The revised text now focuses on describing and interpreting our experimental findings regarding LhERF109's regulatory roles in anthocyanin metabolism without direct figure references. This change maintains the scientific discussion of our results while adhering to the proper format for the discussion section.

Comments 20:

Line 478: this is speculative. For this to be confirmed, knockout or OE lines on the LhERF109 gene should be designed. 

Response:

Thank you for highlighting the speculative nature of our original discussion. We agree that without stable transgenic lines, the functional role of LhERF109 cannot be definitively confirmed. In response, we have revised the discussion and conclusion sections to remove or rephrase statements that may have overstated our findings. Specifically, we removed overinterpretations regarding potential regulatory hierarchies and MBW complex interactions, and now emphasize that our conclusions are based on transient transformation assays, which indicate a possible regulatory role for LhERF109. These changes appear in the revised discussion (lines 470–506), where we have adopted a more cautious and evidence-based tone.

We sincerely thank Reviewer 1 for the comprehensive and thoughtful evaluation of our work. Your comments have guided us to improve the technical accuracy, clarity, and interpretive depth of our manuscript. We have carefully addressed each point and believe that the revised version has benefited substantially from your input. We remain open to further revision if needed and appreciate your valuable contribution to strengthening this study.

Sincerely,

The Authors

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Review of the Article:  Transcriptomic Profiling of Heat-Treated Oriental Lily Reveals  LhERF109 as a Positive Regulator in Anthocyanin Accumulation

 

Summary

The article explores the mechanism underlying tepal color fading in  Oriental lily cultivars under high temperature. It describes the identification of genes by transcriptome analysia and the construction of a gene co-expression network including the key transcription factor LhMYB12 and structural genes involved in the anthocyanin biosynthetic pathway (LhANS, LhDFR, LhUGT78, and LhF3’H).  LhERF109 is identified as a promising regulator of anthocyanin biosynthesis under high temperature, providing new targets for breeding heat-tolerant lilies with stable flower coloration

General Comment

The article presents original results. The experiments are correctly designed and overal it is well written. Figures are OK. Please pay attention to corrections indicated below in the Materials and methods and Results sections.

Comments by Sections

2. Materials and Methods

2.1. Plant Materials and Temperature Treatments

Line 104: Please explain St2-St3 in: plants at developmental St2-St3

2.8. Quantitative Real-time PCR (qRT-PCR)

Line 201: Please explain 2^-△△Ct in: expression level was assessed with 2^-△△Ct method using an internal reference gene LhActin.

3. Results

Figure 1. Explain CK. Explain well sections D and E.

• Gene Expression Differences between NT- and HT-Treated Oriental Lily

Line 235, explain Q30

Line 236, explain N50

3.4. Anthocyanin-Related DEGs Revealed by Analysis of Co-Expression Networks

Is this correct:

 

Figure 5. Weighted gene co-expression network analysis (WGCNA) of DEGs in apple at 20°C

It is correct “apple”. Do these results refeer to apple?

 

Correct:

centered around known anthocyan in biosynthetic genes and identified key …-

to:

centered around known anthocyanin biosynthetic genes and identified key …-

Author Response

Author's response to the reviewer 2

Dear Reviewer 2, 

We sincerely thank the reviewer for the thorough and constructive comments on our manuscript. We have carefully revised the manuscript according to your suggestions. Below are point-by-point responses to each of your comments.

We have carefully revised the manuscript according to your suggestions. All changes made in response to your comments are clearly marked in red in the revised version of the manuscript for your convenience.

Below are our point-by-point responses to each of your comments:

Materials and Methods  

Comments 1:

Line 104: Please explain St2-St3 in: plants at developmental St2-St3

Response:

Thank you for your suggestion to clarify the developmental stages St2–St3. We have now added a detailed explanation in the main text (Line 107), specifying that St2 corresponds to the initial tepal coloration stage and St3 to the partial coloration stage, to improve clarity and reproducibility. Additionally, we have illustrated these stages in Figure 1A and provided a descriptive caption in the figure legend to visually support the textual explanation (Lines 266-268).

Comments 2:

Line 201: Please explain 2^-△△Ct in: expression level was assessed with 2^-△△Ct method using an internal reference gene LhActin.

Response:

Thank you for your comment. As suggested, we have clarified the calculation method for gene expression levels in the revised manuscript (Lines 228–230). Rather than describing the 2^–ΔΔCt method in detail, we now cite the original publication by Livak and Schmittgen, which formally introduced this widely used approach:

48. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^–ΔΔCT method. Methods 2001, 25, 402–408.

Results  

Comments 3:

Figure 1. Explain CK. Explain well sections D and E.

Response:

Thank you for your helpful comment. In the revised manuscript, we have clarified the meaning of "CK" (control, 20 °C) and provided more detailed explanations for panels D and E of Figure 1.  Specifically, we now indicate the types of sugars measured in panel D (soluble sugar, reducing sugar, and sucrose), and panel E displays the qRT-PCR validation results of transcriptome-identified genes, including anthocyanin biosynthetic structural genes and regulatory transcription factors. These changes aim to improve clarity and ensure that the results are clearly interpretable for readers.These revisions appear both in the figure legend and in the main text (Lines 272–278).

Comments 4:

Line 235, explain Q30; Line 236, explain N50

Response:

Thank you for pointing this out. In the revised manuscript, we have added brief explanations for both Q30 and N50 to clarify their significance in evaluating RNA-seq data quality and assembly performance. The revised text now reads (Lines 281–286):

"…generated 143.9 Gb of high-quality clean data, with Q30 > 94.25%, indicating that over 94% of the bases had a Phred quality score ≥ 30 (i.e., a base call accuracy ≥ 99.9%). In the absence of a reference genome for Lilium Oriental, clean reads were de novo assembled using Trinity software, yielding 63,394 Unigenes with an N50 length of 1.58 kb (N50 refers to the contig length such that 50% of the total assembled sequence is contained in contigs of this length or longer)."

These additions improve clarity for readers who may not be familiar with sequencing quality.

Comment 5: 

Figure 5. Weighted gene co-expression network analysis (WGCNA) of DEGs in apple at 20℃-It is correct "apple" . Do these results refeer to apple?

Response:

Thank you for noticing this error. The term "apple" was mistakenly left in the figure legend due to a copyediting oversight. We have now corrected it to "lily" in the revised manuscript (Line 386), as the WGCNA results refer to Lilium (‘Souvenir’) rather than apple. We appreciate your attention to this detail.

Comment 6: 

centered around known anthocyan in biosynthetic genes and identified key …-

Response:

Thank you for your helpful feedback. In the revised manuscript, we have significantly reduced speculative statements in the Discussion section. Specifically, we removed or rephrased overly interpretive comments that lacked direct experimental support—for example, those related to regulatory hierarchies or indirect interactions. The revised discussion now focuses more on the experimental evidence from transcriptome and transient transformation analyses, providing a clearer and more objective interpretation of LhERF109’s potential role in anthocyanin biosynthesis under heat stress. These changes aim to enhance the scientific rigor and avoid unsupported extrapolations.

We sincerely invite the reviewer to examine the revised Discussion section and provide further comments or suggestions. Your insights are highly valuable, and we welcome any additional recommendations to improve the clarity and scientific rigor of our manuscript.

Sincerely,

The Authors

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript aims to characterize the role of LhERF109.  The experimental data seem solid, but the computational analysis is often misinterpreted with overstatements. Extensive rewriting would be necessary for the consideration of publication.   

1. Please, add the correct technical references. Several references are missing including BLAST, Bowtim2, RSEM, KEGG, and WGCNA.
2. Section 3.2: Please, describe how the log2FC was calculated and cite any references if any particular methods were used.
3. Figure 2 and section 3.2: Authors should show some of the raw data either in the form of volcano plot or the heatmap of raw expression of the selected genes. The current visualization does not show the sample-to-sample variability, and many DEGs identified in only one of T1/T2/T3/T4 may not be a real DEG due to sample variability.
4. Figure 3: What is the x scale?
5. Section 3.3 and Figure 4: KEGG pathway analysis seem to highlight very generic stress responses. Please, explain if the p values in Figure 4 (also in Figure 3) are “raw p values” or “adjusted p values”. Conclusions may be quite different depending on which p value was used. 
6. Lines 291-294: There is no mention of ERF109 in the main text, and there is no evidence/data in the paper suggesting that ERF109 is a key regulatory factor. Authors should show its associate GO terms, and the expression patterns over time.  In the current format, ERF109 appears without any contexts.  Also, authors need to show why ERF109 may be more important than other ERF genes.     
7. Section 3.4 and Figure 5: The visualization is poor, and it is impossible to check authors’ claims in this section. At least key genes should be highlighted and legible. 
8. Lines 331-334: This part is a clear overstatement, and there is no evidence to support this (at least in this section). Coexpressed genes do not mean they are coregualted.  Authors need to tone down the results of WGCNA.   

Author Response

Author's response to the reviewer 3

Dear Reviewer 3, 

We sincerely thank Reviewer 3 for the thorough and constructive feedback on our manuscript. We have carefully revised the manuscript in response to your suggestions, which have helped improve the clarity, rigor, and overall presentation of our work. Below, we provide detailed point-by-point responses to each of your comments.

Please note: All modifications in the revised manuscript have been highlighted in red font to facilitate your review.

Materials and Methods  

Comments 1:

Please, add the correct technical references. Several references are missing including BLAST, Bowtie2, RSEM, KEGG, and WGCNA.

Response:

Thank you very much for your helpful comment. In response, we carefully reviewed the Materials and Methods section and have now ensured that all technical tools and databases mentioned in the manuscript are accompanied by appropriate references.

In the revised manuscript, we have added citations for the following tools and methods, and highlighted them in red font for your convenience:

• BLASTx: Altschul et al., 1997 (Nucleic Acids Res. 25, 3389–3402)
• Bowtie2: Langmead and Salzberg, 2012 (Nat. Methods 9, 357–359)
• RSEM: Li and Dewey, 2011 (BMC Bioinformatics 12, 323)
• KEGG pathway analysis: Xie et al., 2011 (Nucleic Acids Res. 39, W316–W322)
• WGCNA: Langfelder and Horvath, 2008 (BMC Bioinformatics 9, 559)

We also took this opportunity to double-check all other software packages, pipelines, and databases used in our study. All of them, including Trimmomatic, Trinity, TransDecoder, DEGseq, Blast2GO, TAIR, MUSCLE, MEGA, JTT, and iTOL, are now properly cited in the revised version (Lines 150-185).

Additionally, we have checked that all gene expression analysis and pathway annotation tools are described consistently and clearly in the Materials and Methods section.

Comments 2:

Please describe how the log₂FC was calculated and cite any references if any particular methods were used.

Response:

We thank the reviewer for this valuable comment. In our study, the log₂ fold change (log₂FC) values were calculated using the DEGseq R package [38], which implements an MA-plot-based method incorporating a random sampling model to estimate expression differences between conditions. The thresholds used for identifying differentially expressed genes (DEGs) were |log₂FC| ≥ 1 and false discovery rate (FDR) < 0.01, as described in Section 2.4 of the Methods.

To improve clarity and consistency, we have revised the corresponding sentence in Section 3.2 of the Results to read (Lines 289-292): 
“Differential expression analysis identified 8,354 DEGs across four pairwise comparisons (|log₂FC| > 1, adjusted p < 0.01), with log₂FC values calculated using the MA-plot-based method in DEGseq.”

38. Wang, L.; Feng, Z.; Wang, X.; Zhang, X. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics 2010, 26, 136–138.

 

Results  

Comments 3:

Figure 2 and section 3.2: Authors should show some of the raw data either in the form of volcano plot or the heatmap of raw expression of the selected genes. The current visualization does not show the sample-to-sample variability, and many DEGs identified in only one of T1/T2/T3/T4 may not be a real DEG due to sample variability.

Response:

Thank you for this valuable comment. We agree that presenting raw expression levels is essential for evaluating biological variation. Accordingly, we revised Figure 5D to show the heatmap based on the FPKM values of all three biological replicates, rather than using average expression. This modification enables visualization of sample-to-sample variability and improves the reliability of the co-expression data (Lines 385).

As for Figure 2, we respectfully chose not to modify it, as it serves to summarize global DEG statistics and time-specific expression trends. Instead, the updated Figure 5D complements this by providing detailed expression patterns of selected genes across replicates.

We have updated the legend of Figure 5D to clarify this revision.

Comments 4:

Figure 3：What is the x scale?

Response:

Thank you for your comment. We have clarified the meaning of the x-axis in the revised legend of Figure 3. Specifically, we have added a sentence to indicate that the x-axis represents the number of differentially expressed genes (DEGs) associated with each enriched GO term. This addition has been made in lines 347 of the revised manuscript.

Comment 5: 

Section 3.3 and Figure 4: KEGG pathway analysis seems to highlight very generic stress responses. Please, explain if the p values in Figure 4 (also in Figure 3) are “raw p values” or “adjusted p values”. Conclusions may be quite different depending on which p value was used.

Response:

Thank you for your insightful comment. In the initial submission, both the GO (Figure 3) and KEGG (Figure 4) enrichment analyses were performed using raw p values. In response to your suggestion, we have re-analyzed both datasets using adjusted p values based on the Benjamini–Hochberg false discovery rate (FDR) correction method.

Accordingly, Figures 3 and 4 have been updated to reflect the adjusted p values, and the corresponding results section (Lines 311–340) has been thoroughly revised. Although the use of corrected p values led to a reduction in the number of significantly enriched terms, the core temporal patterns and biological interpretations remained consistent.

In the GO enrichment analysis, we continued to observe:

• At T1, strong enrichment in classic abiotic stress terms such as ‘response to heat’ and ‘response to reactive oxygen species’, along with membrane-related components;
• At T2, dominant enrichment in ‘RNA modification’, ‘plastid’, and ‘chloroplast’, indicating post-transcriptional regulation and organelle adjustment;
• At T3, terms related to ‘photoprotection’, ‘response to red light’, and developmental regulation;
• At T4, a return to ‘RNA modification’, ‘mitochondrial mRNA processing’, and metabolic regulation, indicating long-term transcriptomic adaptation.

In the KEGG pathway enrichment analysis, similar stability was observed:

• At T1, ‘protein processing in endoplasmic reticulum’ remained the most significantly enriched pathway;
• At T2, moderate enrichment was detected in ‘flavonoid biosynthesis’ and ‘phenylpropanoid biosynthesis’, and ‘plant hormone signal transduction’ appeared for the first time;
• At T3, ‘anthocyanin biosynthesis’ and ‘plant–pathogen interaction’ pathways were detected, aligning with pigment regulation and defense responses;
• At T4, both ‘plant hormone signal transduction’ and ‘plant–pathogen interaction’ showed significant enrichment, along with the appearance of ‘MAPK signaling pathway’ and ‘photosynthesis’.

Overall, while statistical stringency was improved, the main biological conclusions remain unchanged, supporting the robustness of our analysis.

Comment 6: 

Lines 291-294: There is no mention of ERF109 in the main text, and there is no evidence/data in the paper suggesting that ERF109 is a key regulatory factor. Authors should show its associate GO terms, and the expression patterns over time.  In the current format, ERF109 appears without any contexts.  Also, authors need to show why ERF109 may be more important than other ERF genes.    

Response:

Thank you for this insightful comment. In the original submission, we acknowledge that LhERF109 was introduced too early and lacked sufficient biological justification and supporting data. In response, we have removed the sentence that mentioned LhERF109 prematurely in the Results section (Lines 291-294 in the original manuscript).

To address your concerns more appropriately, we have revised the Discussion section to provide a detailed rationale for prioritizing LhERF109. Specifically, we now highlight that LhERF109 was identified from the anthocyanin-associated 'black' WGCNA module and exhibited strong co-expression with known biosynthetic and regulatory genes, including LhMYB12, LhDFR, and LhANS. Furthermore, among the ERF genes within this module, LhERF109 showed the most consistent expression correlation with pigment traits across multiple time points. This pattern, along with its homology to Arabidopsis AtERF109 (a Group X ERF known to mediate hormone crosstalk), led us to prioritize LhERF109 for functional analysis. These changes are presented in the revised Discussion section (Lines 479-496).

In addition, we emphasized that functional validation was conducted via transient overexpression and silencing, with results supporting its role in positively influencing anthocyanin biosynthetic genes. However, we now more cautiously interpret these results as suggestive rather than definitive and acknowledge the need for further studies to confirm direct interactions and mechanisms of action.

We hope these revisions provide better clarity and justification for the selection and interpretation of LhERF109.

Comment 7: 

Section 3.4 and Figure 5: The visualization is poor, and it is impossible to check authors’ claims in this section. At least key genes should be highlighted and legible. 

Response:

Thank you for your helpful feedback. In the revised version of the manuscript, we have replaced Figure 5 with an improved high-resolution version to enhance overall readability. Additionally, we have highlighted key genes, LhERF109 has been distinctly marked with a star symbol (✶) in both the network and heatmap panels to facilitate identification. These changes have improved the clarity of the figure and make it easier for readers to verify the claims made in Section 3.4 (Lines 395 and 398).

Comment 8: 

Lines 331-334: This part is a clear overstatement, and there is no evidence to support this (at least in this section). Co-expressed genes do not mean they are coregulated. Authors need to tone down the results of WGCNA.

Response:

Thank you for this important observation. We fully agree that gene co-expression, as revealed by WGCNA, does not equate to direct co-regulation or functional interaction. The original wording in Lines 331-334 of the previous version may have overstated the implications of our network analysis.

In response to your comment, we have removed the overly strong phrasing and carefully revised the relevant section. The revised text now clearly states that the identified transcription factors are “co-expressed” with anthocyanin-related genes within the WGCNA module, and we now emphasize that these genes are only putative candidates pending further functional validation. This change reflects a more conservative and accurate interpretation of our network analysis results.

And thank you for pointing out the need to avoid speculative conclusions. In the revised manuscript, we have carefully re-examined the Discussion section and made further corrections to avoid overstating our findings. Specifically, we removed or reworded sentences that implied direct regulatory relationships or definitive functional roles for co-expressed genes without experimental validation. For example, the description of LhERF109’s role has been adjusted to reflect its association with anthocyanin-related gene expression rather than direct regulation.

Throughout the revised Discussion (Lines 449-506), we now emphasize the preliminary nature of our observations based on transcriptome co-expression patterns and transient transformation results. We avoid making causal claims and instead frame LhERF109 as a candidate gene worthy of further study, aligning our interpretations with the available data. We appreciate the reviewer’s comment, which helped us strengthen the scientific accuracy of our discussion.

Sincerely,

The Authors

 

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript investigates the response of Lily (Lilium) tepals to temperature changes using transcriptome sequencing and identifies key genes associated with anthocyanin biosynthesis under heat stress. The study finds that high temperatures cause tepal color fading and implicates LhERF109 as a positive regulator in anthocyanin biosynthesis. These findings contribute valuable insights into the gene regulatory network during heat stress and have potential applications in breeding heat-tolerant lily varieties.

While the study is well-structured and provides meaningful results, some revisions are needed before it can be considered for publication. The concerns are detailed below.

Major Comments

1.  Selection of LhERF109 from WGCNA
   • In Section 3.5, the manuscript does not explain how LhERF109 was selected from the WGCNA results. A clear explanation should be provided to justify this selection.
2. Section Title in 3.5
   • The title "phylogenetic tree construction" does not fully cover the content of the section. Consider revising the title to better reflect the overall scope of the section.
3. Figure 6 Issues
   • Bootstrap colors: The figure uses black and red, while the legend indicates green and red. This inconsistency should be corrected.
   • Star and blue circle legends: These markers should be explicitly noted and explained in the figure legend.
   • Lilium ERF sequences retrieval: The methodology for retrieving ERF sequences should be clarified in the Methods section.
   • Number of ERFs in Lilium genome: The manuscript should clarify whether there are only 8 ERF genes in the Lilium genome or if only representative sequences were selected.
   • Implication of LhERF109 similarity with Arabidopsis ERFs: The manuscript states that LhERF109 shows high similarity to Arabidopsis ERFs, but does not discuss the functional implications. Does this suggest a similar regulatory role in lilies? This should be addressed in the Results or Discussion section.

Minor Comments

1. Typos and Formatting

• Line 57 and 59: Malus domestica should be italicized.
• Line 451 and 452: Gene names should follow a consistent italicization format.
• Line 437: "as well as" should not be italicized.

2. Figure and Table Issues

• Line 336 (Figure 5 caption):
  • The phrase "Weighted gene co-expression network analysis (WGCNA) of DEGs in apple" is incorrect.
  • Should be changed to: "in lily" unless the results are indeed from apple.
  • The star mark near ERF109 should be described in the figure legend.
• Figure 7A:
  • A scale bar should be included.
• Figure 1A and 1B:
  • The description should include scale bar length.
• Table 1:
  • The underline in the title should be removed.

3. Gene Names and Terminology

• Full names of genes should be provided at their first appearance. Example:
  • "MBW complex" (Line 471) should be introduced with its full name before using the abbreviation.

4. Image Quality

• The figures in the preprint appear blurry compared to the original images.
• The authors should provide higher resolution images or use a format that preserves resolution after editing.

Author Response

Author's response to the reviewer 4

Dear Reviewer 4, 

We sincerely thank you for your detailed and constructive feedback on our manuscript. Your comments have helped us identify areas that required clarification and improvement. We have carefully revised the manuscript in response to each of your suggestions. All revisions in the main text are highlighted in red for your convenience. Please find our point-by-point responses below:

Major Comments
Comment 1: Selection of LhERF109 from WGCNA

In Section 3.5, the manuscript does not explain how LhERF109 was selected from the WGCNA results. A clear explanation should be provided to justify this selection.

Response:

Thank you for pointing this out. We have revised the manuscript to clarify the rationale for selecting LhERF109 as a candidate transcription factor for further analysis. This selection is supported by the following lines of evidence:

1)WGCNA-based co-expression analysis: LhERF109 was identified within the ‘black’ module, which exhibited the strongest positive correlation with anthocyanin content. It showed high co-expression (R² > 0.95) with multiple structural genes involved in anthocyanin biosynthesis.

2)Functional implication from previous studies: In Malus domestica (apple), MdERF109 has been reported to negatively regulate anthocyanin accumulation, which suggests a potential conserved function in heat-related pigment suppression. Furthermore, its homolog in Arabidopsis thaliana (AtERF109) has been shown to participate in hormone-mediated signaling, including the auxin and jasmonate pathways.

3)Expression pattern under heat stress: In our transcriptomic data, LhERF109 showed strong temperature sensitivity, with its expression nearly undetectable at 35 °C from T2 onward. This expression trend mirrors the suppression of anthocyanin biosynthetic genes under the same conditions.

4)Planned functional validation: While not included in the current manuscript, our preliminary experiments suggest that exogenous application of auxin and methyl jasmonate under high temperature conditions may partially restore floral pigmentation. This observation further supports the potential hormonal involvement of LhERF109, and will be explored in detail in our future functional validation studies.

These explanations have been added to the revised manuscript (Lines 479–496), providing a more comprehensive justification for the selection of LhERF109.

Comments 2: Section Title in 3.5

The title “phylogenetic tree construction” does not fully cover the content of the section. Consider revising the title to better reflect the overall scope of the section.

Response:

Thank you for this helpful suggestion. We agree that the original title did not adequately reflect the full scope of this section, which includes both phylogenetic analysis and functional validation of LhERF109. To address this, we have updated the title to “Phylogenetic and Functional Characterization of LhERF109 as a Positive Regulator of Anthocyanin Biosynthesis”, which better represents the integration of sequence-based and experimental evidence (Lines 399-400).

Comments 3:

Bootstrap colors: The figure uses black and red, while the legend indicates green and red. This inconsistency should be corrected. Star and blue circle legends: These markers should be explicitly noted and explained in the figure legend.

Response:

Thank you for this series of thoughtful suggestions. We have addressed all of these points in the revised manuscript (Lines 433-438):

Bootstrap colors: We have corrected the figure and legend to ensure consistency. The legend now clearly states that black dots represent bootstrap values ≤ 0.5, and red dots represent values between 0.51-1.0.

Star and blue circle legends: These symbols are now explicitly described in the updated legend. Specifically, red stars (★) indicate Lilium ERF genes, and blue circles (●) represent ERF genes from Arabidopsis thaliana.

Comments 4:

Implication of LhERF109 similarity with Arabidopsis ERFs: The manuscript states that LhERF109 shows high similarity to Arabidopsis ERFs, but does not discuss the functional implications. Does this suggest a similar regulatory role in lilies? This should be addressed in the Results or Discussion section.

Response:

Thank you for this insightful suggestion. We have revised the Discussion section (Lines 491-496) to address the potential functional relevance of LhERF109’s phylogenetic relationship with AtERF109. As AtERF109 is known to be involved in hormone-related stress signaling in Arabidopsis, including auxin and jasmonate pathways, we propose that LhERF109 may have a similar regulatory role in lilies.

Although this manuscript does not include direct data on hormone responsiveness, we would like to note that in our follow-up experiments, application of exogenous IAA and MeJA under high-temperature conditions was able to partially restore floral pigmentation, along with an observable increase in LhERF109 expression levels. These findings support the hypothesis that LhERF109 may function at the interface of hormone signaling and pigment regulation.

However, as these results are part of an ongoing study focused on the mechanistic characterization of LhERF109, we have chosen to present them in a separate manuscript. We briefly mention this in the discussion as a promising direction for future research.

Minor Comments

Comments 5: Typos and Formatting

Line 57 and 59: Malus domestica should be italicized；Line 451 and 452: Gene names should follow a consistent italicization format；Line 437: “as well as” should not be italicized.

Response:

Thank you for pointing out these formatting issues. We have carefully reviewed the manuscript and confirm that Malus domestica is now correctly italicized in Line 57 and consistently throughout the text.

Regarding the second and third points, the corresponding sentences have been revised or removed as part of our overall restructuring of the Discussion section. However, we have conducted a thorough check of the entire manuscript and ensured that gene names are italicized consistently and formatting is properly applied. We sincerely appreciate your attention to detail, which has contributed to improving the accuracy and professionalism of the manuscript.

Comment 6: Figure and Table Issues

Line 336 (Figure 5 caption):The phrase "Weighted gene co-expression network analysis (WGCNA) of DEGs in apple" is incorrect,Should be changed to: "in lily" unless the results are indeed from apple.

The star mark near ERF109 should be described in the figure legend

Figure 7A, A scale bar should be included.

Figure 1A and 1B, The description should include scale bar length

Response:

Thank you for these helpful observations. We have carefully revised the manuscript and addressed each of the points:

• The incorrect phrase in the Figure 5 legend has been corrected to “in lily”, as the analysis was conducted on Lilium transcriptomic data (Lines 386).
• The star symbol (✶) marking LhERF109 in Figure 5 has been explicitly described in the updated figure legend (Lines 395 and 398).
• A scale bar has been added to Figure 7A to improve clarity (Lines 439).
• The descriptions of Figures 1A and 1B have been updated to include the scale bar lengths (Lines 268 and 270).

Comment 6: 

Table 1 The underline in the title should be removed.    

Response:

Thank you for pointing this out. The underline in the title of Table 1 has been removed in the revised manuscript to ensure consistent formatting with the rest of the tables and figures.

Comment 7: 

Gene Names and Terminology

Full names of genes should be provided at their first appearance. Example: “MBW complex” (Line 471) should be introduced with its full name before using the abbreviation.

Response:

Thank you for this helpful suggestion. We have revised the manuscript to ensure that the full names of all gene complexes and abbreviations are provided at their first appearance. Specifically, the term “MBW complex” in Line 505 has been updated to read “MYB–bHLH–WD40 (MBW) complex” upon first mention. Similar clarifications have been applied elsewhere in the manuscript where needed. 

Comment 8: 

Image Quality: The figures in the preprint appear blurry compared to the original images.

Response:

Thank you for pointing this out. To address this issue, we have replaced all relevant figures in the revised manuscript with high-resolution versions, and ensured that image formats preserve quality during the editing and submission process. We have also checked each figure carefully to confirm its clarity in the compiled document. We appreciate your feedback, which has helped improve the visual presentation of the manuscript.

Once again, we sincerely thank Reviewer 4 for the detailed and constructive comments. Your feedback has significantly improved the clarity, accuracy, and overall quality of our manuscript. We hope that our revisions have adequately addressed all of your concerns, and we remain grateful for your thoughtful evaluation and suggestions.

Sincerely,

The Authors

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made the appropriate changes according to my suggestions, and clarified doubts I had raised while reading the text. Hence, the quality of the manuscript increased thoroughly. I have listed below some minor adjustments that should bring the manuscript to final publishing quality.

 

- Lines 57-63: The inclusion of these references provided more foundation to the study’s background. However, to improve clarity, it should be explicitly stated how anthocyanin synthetic genes responded in each study. I believe this is necessary since it is the focus of the paper.

- Lines 209-211: Please, include the reference of your group in which this is based upon.

- In some portions of the results onwards, including Fig. 1’s legend, it is mentioned that ‘petals’ were used in the analyses, and not ‘tepals’ as stated in the methods section. Please, revise this carefully.

- Regarding comment 12, I may have mistakenly seen ‘lillies’ when I reviewed the MS, because I could not find it again as well. I apologize for the inconvenience.

- Line 388 – Please, adjust ‘lilium’ term formatting.

- In Figure 6, there is an extra “A” in the upper right corner. If possible, please, delete this extra element in the figure.

- Lines 403-405: the use of the phylogenetic data was well justified in your response. I suggest the inclusion of this justification in the beginning of the text of item 3.5 to improve clarity of why the analyses were made to the readers.

- Line 463: adjust ‘Lilium’ formatting.

Author Response

Author's response to the reviewer 1

Dear Reviewer 1,

We sincerely thank you for your continued review and constructive feedback on our manuscript. Your comments have helped us further refine our work. We have carefully revised the manuscript in response to each of your suggestions. All revisions in the main text are highlighted in red for your convenience. Please find our point-by-point responses below:

Comment 1: Lines 57-63: The inclusion of these references provided more foundation to the study's background. However, to improve clarity, it should be explicitly stated how anthocyanin synthetic genes responded in each study. I believe this is necessary since it is the focus of the paper.

Response: Thank you for your suggestion. In response, we have revised the manuscript to explicitly state how the anthocyanin biosynthetic genes responded in each referenced study. Specifically, we have added the following clarifications (Lines 57-68):

We now explain that MdMYB10 is activated by MdCOL11 under light conditions, promoting the transcription of downstream anthocyanin biosynthesis genes, such as MdDFR and MdUFGT. We also explicitly state that, under high-temperature stress, the expression of MdCOL11 is suppressed, leading to reduced activation of MdMYB10 and impaired transcription of MdDFR and MdUFGT, resulting in decreased anthocyanin accumulation. Additionally, we detail the heat-induced upregulation of MdCOL4, another B-box transcription factor, through heat shock transcription factors (MdHSF3b and MdHSF4a). The MdCOL4-MdHY5 complex formed in response to heat stress represses the expression of genes like MdMYB1, MdANS, and MdUFGT, further inhibiting anthocyanin production.

These additions provide a clearer connection between the studies and the specific response of anthocyanin biosynthetic genes to high-temperature stress.

Comment 2: Lines 209-211: Please include the reference of your group in which this is based upon. 

Response: Thank you for your suggestion. In response to your comment, we have clarified the reasoning behind the selection of the ‘Sorbonne’ cultivar for our transient transformation and VIGS assays. While the ‘Souvenir’ cultivar was used for transcriptome analysis, we chose ‘Sorbonne’ for the functional validation experiments because of its similar pigmentation phenotype and heat-responsive fading behavior. Previous studies have demonstrated that all pink-flowered Oriental lily cultivars, including ‘Sunny Martinique’ [4], ‘Marrero’ [28] and ‘Sorbonne’[47] , exhibit a similar mechanism for heat-induced fading of flower color. Given that ‘Sorbonne’ shows a comparable response to high-temperature-induced color fading and is readily available, it was selected as a practical and representative model for this study (Line 212).

4. Yang, J.; Guo, C.; Chen, F.; Lv, B.; Song, J.; Ning, G.; He, Y.; Lin, J.; He, H.; Yang, Y.; Xiang, F. Heat-induced modulation of flavonoid biosynthesis via a LhMYBC2-Mediated regulatory network in oriental hybrid lily. Plant Physiol. Biochem. 2024, 214, 108966.
5. Lai, Y-S.; Yamagishi, M.; Suzuki, T. Elevated temperature inhibits anthocyanin biosynthesis in the tepals of an Oriental hybrid lily via the suppression of LhMYB12 Sci. Hortic. 2011, 132, 59–65.
6. Lv, S.-Q.; Duan, F.; Zhang, L.-X.; Ning, G.-G.; He, Y.-H. The effect of high-temperature stress on the flower color synthesis of ‘Sorbonne’ lily. J. Huazhong Agric. Univ.2024, 43 (01), 149–156.

 

Comment 3: In some portions of the results onwards, including Fig. 1's legend, it is mentioned that 'petals' were used in the analyses, and not 'tepals' as stated in the methods section. Please, revise this carefully.

Response: Thank you for pointing out this inconsistency. We have carefully reviewed the entire manuscript and standardized the terminology by using "tepals" throughout, as this is the botanically correct term for lily floral organs. We have replaced "petals" with "tepals" in Figure 1 legend (Lines 270，274，276，278 and 281) and in all relevant sections of the results (Lines 245-246).

Comment 4: Line 388 – Please, adjust 'lilium' term formatting.

Response: We have corrected the formatting of "Lilium" to italicized "Lilium" with a capitalized first letter, following standard scientific nomenclature for genus names (Line 391).

Comment 5: In Figure 6, there is an extra "A" in the upper right corner. If possible, please, delete this extra element in the figure.

Response: Thank you for your careful examination. We have removed the extraneous "A" from the upper right corner of Figure 6 as suggested (Line 451).

Comment 6: Lines 403-405: The use of the phylogenetic data was well justified in your response. I suggest the inclusion of this justification in the beginning of the text of item 3.5 to improve clarity of why the analyses were made to the readers.

Response: Thank you for this valuable suggestion. We fully agree that providing a clear justification for the phylogenetic analysis would enhance reader understanding. Following your recommendation, we have added an explanatory paragraph at the beginning of section 3.5 (Lines 407-413) that reads:

"To explore the evolutionary context of LhERF109 and guide our functional studies, a phylogenetic analysis was performed to compare it with ERF family members from Arabidopsis thaliana. Understanding the phylogenetic positioning of transcription factors is crucial for predicting their potential functions and integrating new findings with established knowledge across plant species. This approach is particularly useful for non-model plants, as functional predictions can be made based on well-characterized homologs in model species such as Arabidopsis thaliana." We have also extended our discussion of the phylogenetic results (Lines 416-418) to emphasize their significance: "This close evolutionary relationship provides essential context for interpreting our functional results and forming targeted hypotheses regarding the role of LhERF109 in stress response pathways."

These additions clarify the rationale behind our phylogenetic analysis and its importance for understanding transcription factor function in lily anthocyanin biosynthesis under temperature stress.

Comment 7: Line 463: Adjust 'Lilium' formatting.

Response: We have properly formatted "Lilium" in italics on line 476, consistent with scientific nomenclature standards.

We sincerely appreciate your thorough review and constructive suggestions, which have significantly improved the clarity and quality of our manuscript.

Respectfully submitted,  

All Authors

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has improved, and many of my previous comments were properly addressed.

Author Response

Thank you very much for your thoughtful and constructive feedback. I'm glad to hear that the manuscript has improved and that many of your previous comments have been properly addressed. I appreciate your time and effort in reviewing the manuscript and providing valuable suggestions. I look forward to any further recommendations you may have.

Best regards,

All Authors