Genome-Wide Identification and Expression Analysis of UBP Genes in Peppers (Capsicum annuum L.)

Comments 1: Specify the pepper cultivar ‘6421’ and its relevance to the study. Is it stress-tolerant/sensitive?

Response 1: Thank you for pointing this out. ‘6421’ was used for qRT-PCR and the material used for preparing various samples for RNA sequencing to construct the gene expression database. ‘6421’ has the characteristics of heat resistance, moisture resistance, drought resistance and disease resistance. We have added this information in line 138-140.

Comments 2: The RT-qPCR data for selected genes (CaUBP3/17/27/32/34/35/38) under heat stress and during flower development are insightful. However, the biological replicates and statistical significance should be explicitly stated.

Response 2: Agree. I have completed the revisions to emphasize this point (Line 143-144, 229-233).

Comments 3: The discussion on CaUBP roles in stress response (heat) is speculative without mutant or overexpression studies. Suggest including future directions for functional validation (CRISPR knockouts).

Response 3: Sorry. To further investigate the mechanisms of CaUBP genes under heat stress, subsequent studies could employ gene-editing technologies such as the CRISPR/Cas9 system to knockout or overexpress specific CaUBP genes, enabling direct observation of their functional impact on stress responses.

Comments 4: The subcellular localization prediction (all CaUBPs in the nucleus) seems unusual. Discuss potential limitations of prediction tools or cite experimental validation if available.

Response 4: We fully understand the limitations of subcellular localization prediction tools. These tools typically rely on algorithmic models and training datasets and may have predictive errors. Although we used the commonly utilized plant subcellular localization prediction tool, Plant-mPLoc, to improve prediction accuracy, any predictive results should be interpreted with caution. To address this limitation, we plan to conduct experimental validation in future studies, such as subcellular localization experiments, to confirm the actual localization of CaUBPs. What’s more, we will explore the functional significance of these proteins in the nucleus to further support our prediction results.

Comments 5: The conclusion that CaUBPs regulate flower development is based on expression changes. Address alternative explanations (indirect effects).:

Response 5: I agree with your suggestion and have added this discussion section (Lines 561-568).

Comments 6 Minor grammatical errors ("evironmental stress" / "environmental stress") should be corrected and define abbreviations at first use (HSE, ARE).

Response 6: Thank you for your correction. The term has been revised to "environmental stress" (Line 60).

The abbreviations HSE (heat shock element) and ARE (antioxidant response element) have been spelled out in full at their first occurrence (Line 311, 317).

4. Response to Comments on the Quality of English Language

Point 1: Minor grammatical errors ("evironmental stress" / "environmental stress") should be corrected and define abbreviations at first use (HSE, ARE).

Response 1: Thank you for your correction. The term has been revised to "environmental stress" (Line 60).

The abbreviations HSE (heat shock element) and ARE (antioxidant response element) have been spelled out in full at their first occurrence (Line 311, 317).

Comments 1: L52-L56. Why do the authors focus on the Phyllostachys edulis genes? I think the authors should focus on the close-related species of capsicum. I suggest improve the introduction by adding information of UBP genes in other species related to capsicum.

Response 1: Thank you for your suggestion. I have replaced Phyllostachys edulis with maize (Zea mays) throughout the text. Additionally, the revised manuscript clarifies that no closely related species to pepper (Capsicum annuum) were included in the comparative UBP gene analysis (Line 62-73).

Comments 2: L57. If the previous suggestions is considered. Please, include a conector to the Arabidopsis information.

Response 2: We have adopted your suggestion and incorporated the comparative analysis with Arabidopsis information (Lines 73-75).

Comments 3: L66-L69. Please improve the justification of the study. For example, according to the method section, describe briefly how impact the time and the temperature in these gene family in the flower development. I think this will improve the justification.

Response 3: Thank you for your suggestions. I have improved the justification for the study (Lines 90-95).

Comments 4: Furthermore, add a briefly information about the domain in the introduction section.

Response 4: Thank you for your suggestion. I have added an introduction to the structural domains (Lines 51-57).

Comments 5: L75-L79. All this paragraph, is for the qPCR? For bioinformatic analysis? Please a paragraph or explain the information described here.

Response 5: This description is for performing qPCR analysis.

Comments 6: L82-L89. Please add the date when all the database was consulted.

Response 6: The database accession dates have been added (Lines 147-149).

Comments 7: L134. Please add if a software was use to analyze the protein interaction network. R? Phyton? Tbtools?

Response 7: Sorry, the protein-protein interaction network analysis was conducted using the STRING database, without employing additional standalone software.

Comments 8: L141. Please edit the word “analyse”… analysis… analyses

Response 8: Thank you for your suggestion. I have revised the content accordingly (Line 206).

Comments 9: L142. Add information about how quantify the RNA, and please add the concentration used to create the cDNA.

Response 9: Thank you for your suggestion. We have incorporated this component into the 2.9 RT-qPCR analysis (Lines 208-216).

Comments 10: L145-L151. Based that authors used SYBR green, Do the authors perform a calibration curve? Authors should explain this before the explanation of the reaction mix. Moreover, Please add the concentration of the primers used (10 uM??) and the cDNA used (100 ng)?

Response 10: Thank you for your suggestion. In this study, calibration curves were performed, and the concentrations of both primers and cDNA have now been included (lines 220-227).

Comments 11: L152. Please add the reference for the double double ct method. (Livak, 2001)

Response 11: Thank you for your suggestion. References for the ΔΔCt method have now been included (line 229).

Comments 12: L153-L155. Authors need to specify the experimental design and statistical analysis. Likewise, the information of these lines need to be improved

Response 12: Agree. I have specified both the experimental design and statistical analysis (Lines 220-226).

Comments 13: L327. Substitute hours by h (please review this in the whole manuscript)

Response 13: Thank you for your suggestion. We have standardized all time unit notations to "h" (e.g., 24 h) throughout the manuscript (lines 374, 412).

Comments 14: L328-L336. Authors need to perform a statistical analyses to observe if the are statistical significant differences.

Response 14: Thank you for your suggestions. I have conducted a statistical analysis. Lines 409 - 445.

Comments 15: L340-342. Please add the information of orange line (in the legend of the figure). Indeed, authors should include what is meaning of the lines inside the blue bars… standard deviation? Error standard?

Response 15: Thank you for your suggestions. I have added information about the orange and blue lines in the figure caption. Lines 451 - 453.

Comments 16: L340-342. Based on the figure, could you perform a correlation analysis??

Response 16: I'm sorry, but I'm unable to conduct a correlation analysis.

Comments 17: L347-L351. This information is focused on animals. what is the relation with pepper?

Response 17: Lines L347 to L351 mainly introduce some functions of UBP.

Comments 18: L431. I suggest to include information about a correlation or association with other studies, and improve the discussion with the qPCR results.

Response 18: Thank you for your suggestions. I have added information about the research and improved the discussion of the qPCR results. Lines 548-551.