Review Reports - Coordinated Transcriptional and Metabolic Reprogramming Confers Heat Tolerance in Cucumber

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors comprehensively compared the heat stress responses of cucumber lines with high and low heat tolerance at the transcript and metabolite levels. The data presented in this study could provide valuable foundational information for future crop research focused on heat stress. However, the manuscript requires revision based on the following concerns.

The authors state that the growth of G30 and N24 is comparable under control conditions. However, in the provided images, they appear different: the leaves of G30 seem slightly smaller than those of N24, and their coloration also differs. These variations may indicate differences in fresh weight and pigment levels, even under control conditions, which could influence heat tolerance.

The authors suggest that oxidative damage is a major determinant of heat tolerance between the two lines, based on higher ROS accumulation and MDA levels in G30 compared to N24. However, the ROS and MDA results do not fully correspond to the observed heat tolerance. Although MDA levels are significantly higher in G30 than in N24 at 12 h, differences in leaf damage are already evident after 1 h and 6 h of heat stress. How do the authors explain this discrepancy between MDA accumulation and heat injury?

In Figure 2, DAB and NBT staining images are presented alongside quantitative data for H₂O₂ and O₂⁻ levels. However, the trends in ROS quantification do not align with the staining images. For instance, the graph shows no increase in H₂O₂ levels in G30 at 6 h, whereas the staining image suggests higher accumulation at this time point compared with the control. Similar inconsistencies between images and graphs can be observed throughout Figure 2.

The authors conclude that the maintenance of photosynthesis is important for the higher heat tolerance of N24 compared with G30. This conclusion should be supported by measurements of photosynthetic parameters such as Fv/Fm and photosynthetic rate.

Authors need to carefully check the numbering of Figures. We cannot find Figure 5 in the main text.

Author Response

评论员1

作者全面比较了黄瓜系在转录本和代谢物水平上高低耐热性下的热应激反应。本研究中呈现的数据可能为未来聚焦热应激作物的研究提供宝贵的基础信息。然而，该手稿需要基于以下问题进行修订。

作者指出，在对照条件下，G30和N24的生长是可比的。然而，在提供的图片中，它们看起来不同：G30的叶子似乎比N24略小，颜色也不同。这些变化可能表明新鲜重量和颜料含量存在差异，即使在控制条件下，也可能影响耐热性。

回复：我们真诚感谢评测者的批评。在对照条件下，确实观察到N24和G30叶片大小和颜色上的某些差异，这可能促成了它们固有的（基部）耐热性变化。然而，不同黄瓜基因型之间这种表型差异是不可避免的。为了更准确地区分它们的耐热能力，我们重点研究了高温处理后与耐热性密切相关的生理指标变化，如膜系统、氧化还原稳态和光合作用表现。因此，我们补充了叶绿素荧光数据，以进一步区分它们的应激反应。（参见材料与方法2.3，结果3.1，讨论）

作者认为氧化损伤是两系耐热性的主要决定因素，基于G30中ROS积累和MDA水平高于N24。然而，ROS和MDA结果并不完全符合观测到的耐热性。尽管G30在12小时时MDA水平明显高于N24，但1小时和6小时的热应激后叶片损伤差异已显现。作者如何解释这种多重聚积聚与热损伤之间的差异？

回复：感谢评审者提出这一富有见地的问题。MDA于12小时后被观察到。我们将早期叶片卷曲解读为快速的形态应激反应，而非不可逆热损伤的直接指标。相比之下，ROS的逐步积累和MDA的上升反映了氧化应激压倒细胞防御的临界点，导致膜损伤和后期观察到的严重萎缩表型。因此，早期ROS/MDA积累是最终严重损伤的关键原因，而N24减轻氧化负担的能力是其优越耐热性的主要决定因素。为了澄清这一进程，我们在讨论部分添加了相应的解释和相关参考文献。（参见讨论）

图2中，DAB和NBT染色图像与H₂O₂和O₂⁻水平的定量数据并列呈现。然而，ROS定量的趋势与染色图像不一致。例如，图表显示6小时时G30中的H₂O₂水平没有增加，而染色图像显示该点的H₂O₂积累较对照组更高。图2中也观察到图像与图表之间的类似不一致。

回复：我们真诚感谢评测者的批评。谢谢你的评论。H₂O₂和O₂·⁻的变化代表了一个动态的积累过程。定量测定法与组织化学染色方法之间的差异可能导致结果不完全相同，尽管整体趋势相似。更直接的原因可能是用于定量测量的叶片样本与染色用叶片样本不同，这可能导致个体差异。因此，我们重复了实验处理，重新测试了H₂O₂。更新数据显示，G30中的H₂O₂含量在6小时和12小时均显著增加。（见图2）

作者总结认为，维持光合作用对于N24相较G30更高的耐热性至关重要。这一结论应由光合作用参数如Fv/Fm和光合作用速率的测量支持。

回复：感谢审稿人的建议。谢谢你的建议。我们在手稿中补充了叶绿素荧光参数（Fv/Fm、Fq'/Fm'、qP和ETR）的测量值。相应的数字和数据已添加到结果部分。此外，我们将这些生理发现与讨论中的转录组数据整合，以证实维持光合作用功能对增强N24耐热性的重要性。（参见材料与方法2.3，结果3.1，讨论）

作者需要仔细检查图表的编号。我们在正文中找不到图5。

回复：感谢您提醒我们。在提交系统自动格式化过程中，图5可能被图4无意中叠加。我们已仔细检查并纠正修订稿中的这一问题，以确保图5正确显示。（见图4，图5）

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

3.1 In Figure 1c, they mention a significant difference, but this significance is not evident in the graph (see graph). Why wasn't MDA measured at 48 hours? Why weren't other morphological parameters measured, such as fresh weight, dry weight, etc.?

3.2 I don't know what Figure 4D is. Please provide more details about the genes visualized in the heatmap of Figures 4a and 4b. The reader should be guided, as although some were mentioned in the introduction, they are not given sufficient emphasis. Figure 5 is not mentioned anywhere.

In Table S14, ONLY 12 TRANSCRIPTION FACTORS ARE OBSERVED, not 13 as mentioned in the text. Please review this.

3.3 Figure 6C states that 365 metabolites were overexpressed, while the text mentions 226. Please review this to ensure consistency. Reviewing Tables 1 and 2 and examining the log2FC values, there are no relationships with the most overexpressed factors. The values should be reviewed and the text modified, as the same issue occurs in both cases (N24 and G30). Figure 6D is not mentioned extensively, and the results obtained are not explained.

3.4 Could you mention what types of relationships were observed and what further explanation they might provide?

Discussion: Comparisons with other studies that conducted similar research or observed the same findings are lacking. The references in the discussion only mention factors or genes that have already been described.

Author Response

Response: We thank the reviewer for this insightful question. The significance indicated in Figure 1c is based on statistical analysis of the heat injury index at 48 h, as phenotypic differences between N24 and G30 were most pronounced at this time point. MDA, H₂O₂, and O₂·⁻ were not measured at 48 h because the leaves showed highly heterogeneous damage (including severe wilting or drying) by that stage, which could lead to unreliable or zero readings from severely compromised tissue. We focused on earlier time points (up to 12 h) to capture the progressive accumulation of these oxidative markers, which reflect cellular-level damage even before severe visual symptoms appear. We selected a set of widely reported physiological indicators closely linked to heat stress (membrane stability, ROS, and photosynthetic function). To further support the phenotypic observations, we have supplemented the study with chlorophyll fluorescence measurements, which provide a direct and sensitive assessment of photosynthetic thermotolerance. These data have been added to the Results and discussed accordingly. (see Materials and Methods 2.3, Result 3.1, Discussion)

Response: Thank you for bringing this to our attention. Thank you for pointing out the issues with Figures 4 and 5. In the original submission, a formatting error during typesetting likely caused Figure 5 to be overlaid by Figure 4, resulting in the mislabeling and overlap of the figures. We apologize for any confusion this may have caused. We have carefully corrected the figure arrangement in the revised manuscript. Figure 4 and Figure 5 are now presented separately and correctly labeled. The heatmaps previously referred to as 4a and 4b correspond to the newly separated Figure 5a and 5b, which visualize the expression patterns of key genotype-specific transcription factors (including members of the HSF, bZIP and bHLH families). (see Fig4, Fig5, Result 3.2)

In Table S14, ONLY 12 TRANSCRIPTION FACTORS ARE OBSERVED, not 13 as mentioned in the text. Please review this.

Response: Thank you for pointing out the discrepancy. You are correct. We have carefully reviewed the data and confirmed that 12 transcription factors (not 13) were identified in Table S14. The text in the Results has been updated accordingly. (see Result 3.2)

Response: Thank you for your comment. We have re-examined the relevant data and revised the corresponding description in the Results section to accurately reflect our findings. In addition, we have explicitly stated the metabolite selection criteria (ranking by identification confidence score, with Log₂FC > 1) in the text. We have also added further description and interpretation of the results presented in Figure 6D. (see Result 3.3)

3.4 Could you mention what types of relationships were observed and what further explanation they might provide?

Response: Thank you for this valuable suggestion. Thank you for your comment. In response, we have revised Section 3.4 by adding a more detailed description of the observed relationships and have included further discussion on their potential implications. (see Result 3.4, Discussion)

Response: Thank you for your comment. Thank you for this valuable suggestion. We agree that a broader comparison with related studies enhances the discussion. In the revised manuscript, we have significantly expanded the Discussion section. We now integrate our findings more explicitly with prior research on heat stress responses in cucumber and other crops. (see Discussion)

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Please see attached

Comments for author File: Comments.pdf

Author Response

Response: We have reviewed and corrected the relevant formatting issues. (e.g., italics)

For the “Abstract” section: The text details the findings of this study and provides a brief summary, but does not mention the specific research methods used. It is suggested that this be supplemented.

Response: Thank you for your suggestion. We have revised the Abstract section to include a concise description of the key research methods used. (see Abstract)

Please rearrange keywords in alphabetic order

Response: Thank you for your suggestion. The keywords have been rearranged in alphabetical order.

To make this study complete, I propose adding: 'Hormones whose signal transduction pathways differ between thermotolerant and thermosensitive cucumber

Response: Thank you for pointing out the discrepancy. Thank you for your constructive suggestion. While this study did not directly measure hormone levels under heat stress, our transcriptomic analysis revealed significant enrichment of hormone signaling pathways in the differential responses between the two genotypes. We have incorporated this finding into the revised Discussion section, highlighting the potential role of hormone signaling in shaping thermotolerance. We agree that hormonal profiling would be a valuable direction for future work, and we plan to investigate hormone dynamics in follow-up studies. (see Discussion)

43℃ why did you choose this temperature

Response: Thank you for your question regarding the selection of the treatment temperature. The temperature of 43 °C was chosen based on previous reports in cucumber heat‑tolerance screening studies (references have been added in the Materials and Methods section). In addition, our preliminary experiments indicated that 43 °C effectively differentiated the heat‑tolerance levels of various cucumber genotypes. Therefore, this temperature was selected for the present study to ensure a clear phenotypic and physiological distinction between the heat‑tolerant (N24) and heat‑sensitive (G30) lines. (see Materials and Methods 2.2)

please add measurements of Chlorophyll fluorescence

Response: Thank you for your suggestion. We have supplemented the manuscript with chlorophyll fluorescence measurements (Fv/Fm, Fq’/Fm’, qP, and ETR). The corresponding figures and data have been added to the Results section, along with expanded discussion that integrates these physiological findings with our transcriptomic analysis to further support the distinction in heat tolerance between the two cucumber genotypes. (see Materials and Methods 2.3, Result 3.1, Discussion)

replace the outdated references with more recent and up-to-date ones

+If you could present a summary diagram of the coordinated transcriptional and metabolic reprogramming that confers heat tolerance in cucumber

Response: Thank you for these constructive suggestions. We have revised the manuscript as follows:

1, Updated References: We have carefully reviewed and updated the references throughout the manuscript, particularly in the Introduction and Discussion sections. Outdated references have been replaced with more recent and relevant literature to ensure our discussion is framed within the current state of knowledge.

2, Summary Diagram: As suggested, we have created a summary diagram that illustrates the coordinated transcriptional and metabolic reprogramming proposed to confer heat tolerance in cucumber. This schematic model integrates our key findings on the activation of transcription factors (HSF, bHLH), the upregulation of photosynthesis-related pathways and ABC transporters, and the subsequent role of protective metabolites in alleviating oxidative damage and maintaining photosynthetic efficiency.

(see Introduction, Discussion, Fig9)

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors properly answered the comments and revised the manuscript. The current form of the manuscript is suitable for the publication in this journal.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear all, the work has improved considerably. Thank you very much for accepting the feedback provided.