Exploring MicroRNAs Associated with Pomegranate Pistil Development: An Identification and Analysis Study
, Yu Liu 1,2, Miaomiao Wang 1,2, Jiangli Shi 1,2, Kunxi Zhang 1,2, Pengbo Hao 1,2, Shangwei Song 1,2, Tuanhui Bai 1,2 and Xianbo Zheng 1,2,*Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The work of Zhao and colleagues aims to explore for the first time the landscape of microRNAs (miRNAs) within the reproductive structures of Pomegranate plants. While the aim of the study is of broad scientific interest and the research experimental design appropriate, the manuscript presentation suffers from issues of clarity and coherence.
In general, I found confusing what development process the authors want to focus on. Sometimes they mention flowers, other times pistils or ovules. However since smallRNA-seq analysis was conducted on pistils, why not consistently refer to pistils throughout the manuscript? There is a lack of clarity regarding the morphological and functional differences of pistils in hermaphroditic flowers compared to male flowers. Do the pistils of male flowers form ovules at all or do they form defective ones or do they form ovules then abort later as mentioned in lines 209-210? This aspect is crucial for interpreting the data regarding differentially expressed miRNAs that could have a specific function in this developmental process. The authors reference a previous work by Zhao et al. 2023, but for clarity, I believe morphological images of the different flowers/pistils at the three different stages, or at least a diagram/scheme, would assist the reader in comprehension.
Here are my other suggestions to enhance the overall readability and accessibility of the scientific content.
- The Abstract required extensive editing of the English language.
- The introduction focuses on miRNAs in general and a description of Pomegranate reproductive development. All the information about other miRNAs involved in Arabidopsis flower development can be kept for the discussion section. Reviews regarding small RNAs in reproductive development could be mentioned (Petrella et al., 2021 The emerging role of small RNAs in ovule development, a kind of magic. Plant Reprod. doi: 10.1007/s00497-021-00421-4.and Nag A, Jack T. Sculpting the flower; the role of microRNAs in flower development. Curr Top Dev Biol. 2010. doi: 10.1016/S0070-2153(10)91012-0.)
- Lines 176 to 184 are very difficult to follow.
- Regarding the target genes mentioned in Table 4, it would be useful to mention if those targets belong all to the same family or a ’new targets’. For instance, the targets mentioned for mir160 are all genes that encode for ARF transcription factors? This information should be provided for at least the miRNAs and targets that are then analyzed more in detail such as mir158, mir172, and mir160.
- The RNA-seq data have been done in this work? Because information regarding RNA-seq is not reported in material and methods.
- The GO analysis reported in Figure 5 it is not very useful as it reports categories that are too generic.
- The quality of Figure 8 is low and almost not readable. Is that qRT-PCR stem-loop PCR that detects mature miRNAs or qRT-PCR on pri-miRNA precursor? Which normalizer/housekeeping gene has been used? Why legend of Figure 8 mention ‘bud vertical diameter’ instead of pistil length?
Comments on the Quality of English Language
Extensive editing of the English language is required, of Abstract in particular.
Author Response
Dear Editors,
The authors would like to thank all the reviewers for their time and effort in improving this manuscript. The following is a point-by-point response to each comment of all two reviewers.
Sincerely,
Yujie Zhao
Reviewer #1 (Comments for the Author):
The work of Zhao and colleagues aims to explore for the first time the landscape of microRNAs (miRNAs) within the reproductive structures of Pomegranate plants. While the aim of the study is of broad scientific interest and the research experimental design appropriate, the manuscript presentation suffers from issues of clarity and coherence.
In general, I found confusing what development process the authors want to focus on. Sometimes they mention flowers, other times pistils or ovules. However since smallRNA-seq analysis was conducted on pistil, why not consistently refer to pistils throughout the manuscript? There is a lack of clarity regarding the morphological and functional differences of pistils in hermaphroditic flowers compared to male flowers. Do the pistils of male flowers form ovules at all or do they form defective ones or do they form ovules then abort later as mentioned in lines 209-210? This aspect is crucial for interpreting the data regarding differentially expressed miRNAs that could have a specific function in this developmental process. The authors reference a previous work by Zhao et al. 2023, but for clarity, I believe morphological images of the different flowers/pistils at the three different stages, or at least a diagram/scheme, would assist the reader in comprehension.
Response: Thanks to reviewer for their comments. We have changed and used ‘pistil’ in the manuscript.
Pomegranate trees produce large numbers of both bisexual flowers that produce fruit and functional male flowers that typically drop and fail to set fruit. Bisexual flowers have the discoid stigma covered with copious exudate, elongated stigmatic papillae, the single elongate style, numerous and anatropous ovules. In contrast, functional male flowers have reduced female parts and exhibited shortened pistils of variable heights. Functional male flowers have sterile pistils that show abnormal ovule development. The integument primordia formed and then ceased developing in the ovules of functional male flowers with a vertical diameter of 8.1–13.0 mm (Zhao yujie et al., 2023). Relevant information have been added to at the introduction of article (Line 83- Line 86).
Morphology of flowers and ovules in bisexual and functional male flowers of pomegranate have been presented in Zhao et al (2021) and Zhao et al (2023). and if the pictures are added to this article, there will be a problem of reuse, so this article references our published article as the basis.
Yujie Zhao, et al. 2021. Cloning and Spatiotemporal Expression Analysis of PgWUS and PgBEL1 in Punica granatum[J]. Acta Horticulturae Sinica, 48(2): 355-366. (in Chinese)
YuJie Zhao, et al. BELL1 interacts with CRABS CLAW and INNER NO OUTER to regulate ovule and seed development in pomegranate. Plant Physiology 2023, 191, 1066-1083.
Here are my other suggestions to enhance the overall readability and accessibility of the scientific content.
1、The Abstract required extensive editing of the English language.
Response 1: Thanks to reviewer for the comment. We have revised and checked the file.
2、The introduction focuses on miRNAs in general and a description of Pomegranate reproductive development. All the information about other miRNAs involved in Arabidopsis flower development can be kept for the discussion section. Reviews regarding small RNAs in reproductive development could be mentioned (Petrella et al., 2021 The emerging role of small RNAs in ovule development, a kind of magic. Plant Reprod. doi: 10.1007/s00497-021-00421-4.and Nag A, Jack T. Sculpting the flower; the role of microRNAs in flower development. Curr Top Dev Biol. 2010. doi: 10.1016/S0070-2153(10)91012-0.)
Response 2: We have already referred to this article (Line 60).
3、Lines 176 to 184 are very difficult to follow.
Response 3: Thanks to reviewer for the comment. We have revised and checked the file.
4、Regarding the target genes mentioned in Table 4, it would be useful to mention if those targets belong all to the same family or a ’new targets’. For instance, the targets mentioned for mir160 are all genes that encode for ARF transcription factors? This information should be provided for at least the miRNAs and targets that are then analyzed more in detail such as mir158, mir172, and mir160.
Response 4: Thanks to reviewer for the comment. We have revised ‘3.4 miRNA target gene prediction’.
5、The RNA-seq data have been done in this work? Because information regarding RNA-seq is not reported in material and methods.
Response 5: Thanks to reviewer for the comment. The RNA-seq data have been published in our previous articles (Zhao et al., 2023) (Line 157).
Zhao, Y.J., Wang, Y.Y., Yan, M., et al. BELL1 interacts with CRABS CLAW and INNER NO OUTER to regulate ovule and seed development in pomegranate. Plant Physiology 2023, 191, 1066-1083.
6、The GO analysis reported in Figure 5 it is not very useful as it reports categories that are too generic.
Response 6: Thanks to reviewer for the comment. Figure 5 was included in Supplementary data (Supplemental Figure 1).
7、The quality of Figure 8 is low and almost not readable. Is that qRT-PCR stem-loop PCR that detects mature miRNAs or qRT-PCR on pri-miRNA precursor? Which normalizer/housekeeping gene has been used? Why legend of Figure 8 mention ‘bud vertical diameter’ instead of pistil length?
Response 7: Thanks to reviewer for the comment. Mature miRNAs were detected using qRT-PCR, PgActin as normalizer gene has been used. We have revised ‘2.2.7 qRT-PCR verification of sequencing results’.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
Dear editors and authors, I have read and reviewed the manuscript entitled Exploring MicroRNAs Associated with Pomegranate Ovule Development: An Identification and Analysis Study, authored by Yujie Zhao, Jingyi Huang, Ming Li, Hongfang Ren, Jian Jiao, Ran Wan, Yu Liu, Miaomiao Wang, Jiangli Shi, Kunxi Zhang, Pengbo Hao, Shangwei Song, Tuanhui Bai and Xianbo Zheng. This manuscript intends to identify miRNAs which play the important role in the ovule development of bisexual flowers and functional male flowers from pomegranate by mRNA sequencing analysis.
The following are some suggestions for further revisions:
1. Even the manuscript describes several miRNAs involved in ovules development, it is strongly suggested that authors propose a model for regulation of flowers development in pomegranate through interactions between identified miRNA and target genes over its different stages.
2. The quality of figure 7 can be improved. Also, that figure is not adequately described nor discussed.
3. Line 301: Please make the correction of the word "suare".
4. Tables 1 and 3 could be included in the complementary section.
5. In the conclusion section, please emphasize the correlation between the identified miRNAs and the stages of ovule development.
6. The text must be thoroughly edited for its language and style in English.
Comments for author File: Comments.pdf
Comments on the Quality of English Language
Author Response
Dear Editors,
The authors would like to thank all the reviewers for their time and effort in improving this manuscript. The following is a point-by-point response to each comment of all two reviewers.
Sincerely,
Yujie Zhao
Reviewer #2 (Comments for the Author):
Dear editors and authors, I have read and reviewed the manuscript entitled Exploring MicroRNAs Associated with Pomegranate Ovule Development: An Identification and Analysis Study, authored by Yujie Zhao, Jingyi Huang, Ming Li, Hongfang Ren, Jian Jiao, Ran Wan, Yu Liu, Miaomiao Wang, Jiangli Shi, Kunxi Zhang, Pengbo Hao, Shangwei Song, Tuanhui Bai and Xianbo Zheng. This manuscript intends to identify miRNAs which play the important role in the ovule development of bisexual flowers and functional male flowers from pomegranate by mRNA sequencing analysis.
The following are some suggestions for further revisions:
Minor comments:
1、Even the manuscript describes several miRNAs involved in ovules development, it is strongly suggested that authors propose a model for regulation of flowers development in pomegranate through interactions between identified miRNA and target genes over its different stages.
Response 1: Thanks to reviewer for the comment. We are also trying to construct a developmental regulatory model. If the model is built on the basis of the verified relationship between miRNA and target genes, it is more convincing, which needs to be confirmed by more in-depth studies. However, this paper focuses on miRNA sequencing, so your suggestions will be carried out in our next step.
2、The quality of figure 7 can be improved. Also, that figure is not adequately described nor discussed.
Response 2: Thanks to reviewer for the comment. We have revised and checked the file.
3、Line 301: Please make the correction of the word "suare".
Response 3: Thanks to reviewer. We have changed ‘suare’ to ‘square’.
4、Tables 1 and 3 could be included in the complementary section.
Response 4: Thanks to reviewer for the comment. Tables 1 and 3 were included in Supplementary data (Supplemental Table 1 and Supplemental Table 2).
5、In the conclusion section, please emphasize the correlation between the identified miRNAs and the stages of ovule development.
Response 5: Thanks to reviewer for the comment. We have revised and checked the conclusion.
6、The text must be thoroughly edited for its language and style in English.
Response 6: Thanks to reviewer for the comment. We have revised and checked the file.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors
Line 190: Should move reference to 2. Fig. currently reading the description it is not clear which image the text refers to. Explanations (lines 218-226, 227-235, 334-339) under Fig.3, Fig.4, Fig. 5 and Fig. 8, are difficult to understand and unexpressive, the textual analysis is too short, the reader needs a lot of time to understand the pictures.
It would be recommended to rework the discussion, in the current version it is too short and unexpressive and only partially reveals the importance and novelty of the study.
Author Response
Dear Editors,
The authors would like to thank all the reviewers for their time and effort in improving this manuscript. The following is a point-by-point response to each comment of all two reviewers.
Sincerely,
Yujie Zhao
Reviewer #3 (Comments for the Author):
1、Line 190: Should move reference to 2. Fig. currently reading the description it is not clear which image the text refers to. Explanations (lines 218-226, 227-235, 334-339) under Fig.3, Fig.4, Fig. 5 and Fig. 8, are difficult to understand and unexpressive, the textual analysis is too short, the reader needs a lot of time to understand the pictures.
Response 1: Thanks to reviewer for the comment. We have revised and detailly explained the naming of materials in 2.1 Plant Materials.
2、It would be recommended to rework the discussion, in the current version it is too short and unexpressive and only partially reveals the importance and novelty of the study.
Response 2: Thanks to reviewer for the comment. We have revised the discussion.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
I would like to express my gratitude to the authors for incorporating all of my suggestions. I believe that the manuscript has significantly improved following the revisions, particularly in the introduction and discussion sections. The additional detailed information provided in the 'Plant Material' section enhances readers' comprehension of the developmental context and morphological distinctions between bisexual flowers and functional male flowers, even in the absence of accompanying images.
Nevertheless, I still have a few minor concerns:
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Please consider rephrasing lines 508-510.
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In paragraph 3.6 (lines 621-645), if you are referring to mature miRNAs, I suggest writing them in roman typeface rather than italics (e.g., PgmiR159a, Pgnovel178).
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In the Materials and Methods section 2.2.7 (qRT-PCR Verification of Sequencing Results), there is a lack of crucial information. Could you specify which samples were utilized for the analysis? Were the same total RNA samples used for sequencing? Additionally, please provide details on the protocol used for qRT-PCR—specifically, whether it involved SYBR Green or probes. I observed that the primers used in the experiments contain a portion of identical nucleotides followed by miR-specific sequence. Could you please describe the primer design? Furthermore, I believe that, in this type of qRT-PCR targeting mature miRNAs, actin might not be the most suitable normalizer. Perhaps a small nuclear RNA (snRNA) would be a better choice.
Comments on the Quality of English Language
Minor editing of English language required
Author Response
Reviewer #1 (Comments for the Author):
1、In paragraph 3.6 (lines 621-645), if you are referring to mature miRNAs, I suggest writing them in roman typeface rather than italics (e.g., PgmiR159a, Pgnovel178).
Response 1: Thanks to reviewer for the comment. We have revised and checked the file.
2、In the Materials and Methods section 2.2.7 (qRT-PCR Verification of Sequencing Results), there is a lack of crucial information. Could you specify which samples were utilized for the analysis? Were the same total RNA samples used for sequencing? Additionally, please provide details on the protocol used for qRT-PCR—specifically, whether it involved SYBR Green or probes. I observed that the primers used in the experiments contain a portion of identical nucleotides followed by miR-specific sequence. Could you please describe the primer design? Furthermore, I believe that, in this type of qRT-PCR targeting mature miRNAs, actin might not be the most suitable normalizer. Perhaps a small nuclear RNA (snRNA) would be a better choice.
Response 2: Thanks to reviewer for the comment. The remaining RNA from miRNA sequencing was used for qRT-PCR. Reverse transcription was performed using the PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa). The primer was designed and synthesized according to the report of Chen et al. (2005). After the primers were mixed, the temperature was set according to the hybrid reverse transcription method introduced by Tang et al. (2006).
qRT-PCR was performed using SYBR® Premix Ex TAQTⅱ (Tli RNaseH Plus) (TaKaRa). The test was carried out according to the kit instructions. Finally, The PCRs were performed on the Applied Biosystems 7500 and the thermal cycler was set as follows: pre-denaturation at 95℃ for 30 s, denaturation at 95℃ for 5 s, and denaturation at 60℃ for 34 s for 40 cycles, and fluorescence was acquired at the second step of each cycle; dissolution curve was gained as follows: 95℃ for 15 s, 60℃ for 60 s and 95℃ for 15 s. Three biological and technical replicates were designed for each miRNA.
We quite agree with your opinion, that snRNA is used as the normalizer gene. In this experiment, U6 was first considered as the normalizer gene, but the same normalizer gene was used to consider the consistency with the transcriptome sequencing, so PgActin as normalizer gene had been used.
Chen C., Ridzon D.A., Broomer A.J., Zhou Z., Lee D.H., Nguyen J.T., Barbisin M., Xu N.L., Mahuvakar V.R., Andersen M.R., Lao K.Q., Livak K.J., Guegler K.J. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Research, 2005, 33(20): e179-e179.
Tang F., Hajkova P., Barton S.C., Lao K., Surani M.A. MicroRNA expression profiling of single whole embryonic stem cells. Nucleic Acids Research, 2006. 34(2): e9.
Author Response File: Author Response.pdf
