PavSPL Expression Dynamics in Fruits and Seeds and in Relation to Endocarp Lignification Status During the Transition from Development to Ripening in Sweet Cherry

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments and Suggestions for manuscript horticulturae-3641289 “PavSPL expression dynamics in fruits and seeds and in relation to endocarp lignification status during the transition from development to ripening in sweet cherry” to the authors.

The authors of manuscript horticulturae-3641289 present a comprehensive analysis of PavSPL gene expression dynamics associated with endocarp lignification during fruit development in sweet cherry. The study effectively utilizes two contrasting cultivars—‘Celeste’ (early-season) and ‘Regina’ (late-season)—to investigate differences in fruit and seed development, lignification, and pigmentation.

The experimental design is robust, and the analyses are thorough and clearly explained in the results section. The discussion effectively contextualizes the findings with relevant published literature. The results demonstrate a strong association between endocarp lignification, color development, and the expression of miR156-targeted PavSPL transcription factors. Specifically, early-season 'Celeste' exhibits earlier lignin deposition, pigment accumulation, and elevated PavSPL expression compared to 'Regina'. Transcriptomic and co-expression analyses highlight PavSPL2 and PavSPL9 as key regulators of lignin and anthocyanin biosynthesis. Moreover, auxin-related treatments show potential to modulate PavSPL expression, reduce lignification, and enhance pigmentation.

These findings offer valuable insights into the hormonal and genetic regulation of ripening in sweet cherry, providing promising PavSPLs genes for future sweet cherry breeding programs for improving fruit quality, pigmentation, and ripening time.

I recommend the manuscript for acceptance with minor revisions, as outlined below.

Specific Comments

Abstract:

• Consider adding 4–6 keywords to enhance discoverability and help readers better understand the focus of the study.

2. Materials and Methods

2.1 Plant Material:

• The manuscript mentions the use of three sweet cherry cultivars namely Celeste’ Regina, and Lapins’ However, the biological status of these cultivars (e.g., wild types, landraces, or breeding lines/registered varieties) is not provided. Please include this information for each cultivar.
• Additionally, specify the source of the plant material, whether these cultivars were obtained from a gene bank, breeding program, or research institute.
• The cultivar ‘Lapins’ should be clearly introduced in this section, as it plays an important role in the experimental design experiment in year 2023-24 abd 2024-25.

2.2 Experimental Design:

• The rationale behind using four treatments in the 2023–2024 experiment (Section 2.2.2) and five treatments in the 2024–2025 experiment (Section 2.2.3) is not clearly explained. Please clarify the difference in treatment numbers and experimental purpose across the two years for improved reader understanding.

5. Conclusion:

• The conclusion section is currently very brief. I recommend expanding it to include key take-home messages and practical implications of the findings, particularly the role of PavSPL genes and their potential use in sweet cherry breeding strategies.

Author Response

Reviewer #1

I recommend the manuscript for acceptance with minor revisions, as outlined below.

We thanks Reviewer #1 for reviewing this article in detail.

 

Specific Comments:

1. Abstract: Consider adding 4–6 keywords to enhance discoverability and help readers better understand the focus of the study.

Response 1: Thank you for the observation. The keyword are demanded we the article is submitted to MDPI and not part of the main text to my knowledge.

2. Materials and Methods

2.1 Plant Material:

The manuscript mentions the use of three sweet cherry cultivars namely Celeste’ Regina, and Lapins’ However, the biological status of these cultivars (e.g., wild types, landraces, or breeding lines/registered varieties) is not provided. Please include this information for each cultivar.

Response 2.1: Thanks for noting this. These cultivars are registered varieties. This was clarified in the text.

Additionally, specify the source of the plant material, whether these cultivars were obtained from a gene bank, breeding program, or research institute.

The cultivar ‘Lapins’ should be clearly introduced in this section, as it plays an important role in the experimental design experiment in year 2023-24 abd 2024-25.

We thanks Reviewer #1 for the observation. The plant material was commercially obtained, accompanied by a certificate indicating the sweet cherry variety. This is in line 170.

2.2 Experimental Design:

The rationale behind using four treatments in the 2023–2024 experiment (Section 2.2.2) and five treatments in the 2024–2025 experiment (Section 2.2.3) is not clearly explained. Please clarify the difference in treatment numbers and experimental purpose across the two years for improved reader understanding.

Response 2.2: The point indicated by the reviewer regarding the repetition in the 2024-2025 season was explained in the Results section: “The effect of p-IBA and auxin treatments on lignification was assessed in the following season (2024-2025) in the same cultivar (Fig. 7). The NAA analog was used instead of IAA and both compounds, p-IBA and NAA, were tested at two doses.”

Also in Discussion section the sentence “Finally, field not controlled conditions could influence the treatment effect. Therefore, we repeated the assay the following season, 2024–2025, by focusing on treatments that improved color and with two concentrations.” also justifies the five treatments.

Nevertheless, for further clarification, we have added the following sentence to the Results section in the revised version: “Thus, the five treatments further explored the effect of treatments that produced significant differences in the 2023-2024 season”, lines 487-489.

3. Conclusion:

Response 3. The conclusion section is currently very brief. I recommend expanding it to include key take-home messages and practical implications of the findings, particularly the role of PavSPL genes and their potential use in sweet cherry breeding strategies.

We thanks Reviewer #1 for the suggestion, which will help improve the reach of our work. We added the following sentences in the Conclusion section: “These treatments revealed that the endocarp lignification process is relevant to the transition to ripening. Future studies should further explore the effects of compounds that disrupt lignification, with the aim of developing growth regulators useful for orchard management. Finally, the SPL/miR156 module is a promising candidate for identifying genetic factors that could be used in marker-assisted breeding programs”. This is in lines 724-729 of the new version.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript investigates the expression dynamics of PavSPL genes in sweet cherry fruits and seeds and their association with exocarp lignification, which holds scientific relevance for Chile’s cherry industry. However, the following issues must be addressed before further consideration:

1. Line numbers are absent in the current manuscript, complicating the review process.
2. The abstract’s background section (first eight lines) is excessively lengthy and should be condensed to two concise sentencesor less.
3. The Introduction section requires restructuring: The rationale for selecting SPLgenes as the research focus must be clarified by first introducing the biological significance of SPL genes before expanding on miR156s.
4. In Figure 1C, significance markers for t-test results should be represented with asterisks (*, **, ***).
5. Figures 2B and 2C lack statistical significance indicators (e.g., t-test) for the presented data comparisons.
6. Figure 3 suffers from poor visual clarity and requires enhanced resolution or labeling.
7. Table 1 is incomplete and must be reformatted as a standard three-line table.
8. The one-way ANOVA labeling in Figures 6D and 7B is incorrect. Values should follow descending alphabetical order (e.g., larger values labeled “a,” smaller values labeled “b”).

Substantial revisions are required to align this manuscript with the journal’s publication standards.

 

Author Response

Reviewer #2

This manuscript investigates the expression dynamics of PavSPL genes in sweet cherry fruits and seeds and their association with exocarp lignification, which holds scientific relevance for Chile’s cherry industry. However, the following issues must be addressed before further consideration:

We Thanks to Reviewer #2 for reviewing this article in detail.

1. Line numbers are absent in the current manuscript, complicating the review process.

Response 1: Thanks, we requested the editorial team to generate a new version with the line numbers.

2. The abstract’s background section (first eight lines) is excessively lengthy and should be condensed to two concise sentences or less.

Response 2: We shortened that part to describe the background in a more concise way. We deleted sentences in lines 14 and 20.     

3. The Introduction section requires restructuring: The rationale for selecting SPLgenes as the research focus must be clarified by first introducing the biological significance of SPL genes before expanding on miR156s.

Response 3: We thank the reviewer for pointing out the need to improve the clarity of the introduction. This was a valuable observation to articulate a key conceptual aspect of our study. SPL genes do not have biological significance on their own, but rather as part of the SPL/miR156 regulatory module. This is because in the literature, when a SPL is present and governs a process, a miRNA156 regulates its expression. Therefore, it is difficult to separate SPLs and miR156 as independent elements, since most studies investigate SPL genes in conjunction with their corresponding miRNA156. In our case, although we focused primarily on SPLs, we also attempted to detect miR156 and determine its putative target alignment with our SPLs. Overall, it is conceptually more appropriate to treat SPL and miR156 as a unified functional module. Accordingly, in the Introduction section, the role of SPL genes must be followed by miR156 description. Nevertheless, we have taken the reviewer’s suggestion into account and revised the introduction to make this point clearer, indicating the existence of this regulatory module (line 101 of the new version)

4. In Figure 1C, significance markers for t-test results should be represented with asterisks (*, **, ***).

Response 4: That is correct. Thanks for noting this. Already fixed.

5. Figures 2B and 2C lack statistical significance indicators (e.g., t-test) for the presented data comparisons.

Response 5: That is correct. Thanks for noting this. Already fixed.

6. Figure 3 suffers from poor visual clarity and requires enhanced resolution or labeling.

Response 6: We resized the figure for better visualization.

7. Table 1 is incomplete and must be reformatted as a standard three-line table.

Response 7: Table 1 was modified to include the additional information retrieved from the psRNATarget tool, as requested. However, we believe the data would be better visualized in a vertical orientation, which depends on Horticulturae formatting guidelines. The UPE parameter was not calculated by the tool in this analysis, so default placeholder values were retrieved and therefore excluded from the final table. The start and end positions of the miRNA were also omitted, as this information is considered self-evident. In contrast, the position on the targets and the expectation score were retained, as it reflects the reliability of the predicted inhibitory interaction. In addition, the table layout was adjusted to follow a standard format, with all rows clearly marked for consistency.

8. The one-way ANOVA labeling in Figures 6D and 7B is incorrect. Values should follow descending alphabetical order (e.g., larger values labeled “a,” smaller values labeled “b”).

Response 8. We adhered to the output provided by the InfoStat software for Figures 6D and 7B. For instance, the chlorophyll-related analysis in Figure 6D was labeled according to the post-ANOVA groupings automatically generated by the software.

The image for illustrating this is in the PDF document "Response to Reviewers Horticulturae"    

Accordingly, the letters indicating statistical differences were maintained in the same order in the final figures. However, if you still consider that the order should be adjusted for clarity or consistency, we are open to modifying the lettering in the figures.

Author Response File: Author Response.pdf