Transcriptomic Analysis of Alfalfa Flowering and the Dual Roles of MsAP1 in Floral Organ Identity and Flowering Time

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General Comments:

• Italicize Gene Names: Please use italics for all gene names throughout the manuscript to ensure consistency and adhere to standard scientific formatting.

• Update Citations: It is recommended to include citations to newly published articles in 2024, particularly in the discussion section, to ensure the manuscript references the most current research.

• Software and Tools References: In the Materials and Methods section, ensure that all software, packages, and tools used are properly referenced.

• Conclusion Content: The conclusion should summarize the key results of your study rather than providing only general statements.

Specific Comments:

• P1, L11: Replace "Day 25" and "Day 35" with the corresponding growth stages. Detailed timings should be mentioned in the Materials and Methods section.

• P1, L22: Change "MsAP1" to MsAP1 in italic to follow the correct format for gene names.

• P2, L71: Change "MsAP1" to MsAP1.

• P2, L83: You mentioned "Nicotiana benthamiana" but did not provide further information in the results and discussion sections. Please include additional details or remove the reference if not relevant.

• P2, L87: Replace "The plant apical ..." with "The alfalfa plant apical ...".

• P2, L95: Trinity is primarily used for de novo RNA-Seq transcriptome assembly. If you used it for reference-based analysis, please justify this choice, possibly indicating if the alfalfa genome reference is of low quality or fragmented.

• P3, L106: Provide more details about the parameters used with the "clusterProfiler" package for GO analysis.

• P3, L108: Include the primer list for RT-qPCR in the supplementary file and reference this in the main text.

• P3, L110: If you used a kit, include the catalog number (Cat Number).

• P3, L113: The alfalfa gene format "MS.gene013348" is not readily found online. Mention if there is a distinct database for alfalfa genes and reference it in the Materials and Methods section.

• P3, L118: Correct the term "coned" to "cloned".

• P3, L125: Correct "4mg /L" to "4 mg/L".

• P3, L129: Change "build the e neighbor-joining" to "build the neighbor-joining".

• P4, L144: Correct "students` t - test" to "student's t-test".

• P4, L145: It is preferred to use the LSD test instead of the Duncan test for statistical analysis.

• P4, L164-5: Replace 3.66% and 9.3% with 28.22% (962/4309) and 71.78% (2447/3409), respectively, for clarity and precision.

• P5, L170: Specify if the R²=0.7679 in Fig1f is significant.

• P6, L194: In Fig2d, specify the three parts of each SA and FB in the figure for clarity.

• P7, L235: In Fig3a, provide the full names of the abbreviated gene names (e.g., LFY, FUL) in the figure caption. When these names appear in the text for the first time, they should not be abbreviated.

By addressing these general and specific comments, your manuscript will be clearer, more accurate, and better aligned with scientific standards.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Flowering is an important process for plants as well as crop species. To explore the transcriptomic profile of alfalfa flowering transition, the authors compared gene expression between shoot apices (SA) at Day 25 and flower buds (FB) at Day 35 by mRNA sequencing. They found that 79.1% DEGs showed higher expression level in SA than FB, suggesting they were sequentially turned on and off at the two test stages. For the DEGs encoding the components of circadian rhythm, sugar metabolism, phytohormone signaling, and floral organ identity genes, 60.71% showed higher abundance in FB than SA.

Among them, the authors focused in MsAP1, an APETALA1 (AP1) homolog of Arabidopsis. They performed ectopic expression study of MsAP1 in Arabidopsis resulted in dwarfism and early flowering under the long-day conditions. Furthermore, the authors revealed that MsAP1 is localized in the nucleus and exhibits significant transcriptional activity.

The authors found that a transcriptional regulation network of alfalfa transition from juvenile phase to flowering and provided genetic evidence of the dual role of MsAP1 in flowering and floral organ development.

1. The transcriptome analysis was well done, and the results of the analysis seem to have produced certain new scientific findings. However, the network analysis in Fig. 3b is not described in detail, which is unkind. Since this paper compares SAM and flower buds and concludes that the MsAP1 gene has a dual function, a detailed explanation and discussion of the network analysis should be provided.

2. It is important to recognize that the Arabidopsis transformation experiments cannot say anything about the function of MsAP1 in alfalfa. If MsAP1 is overexpressed in Arabidopsis, we only know about the effects of excess of this protein on the Arabidopsis phytoplasma, but not what happens to the alfalfa phytoplasma. To determine the function of a gene in a given plant, it is necessary to analyze mutants of that gene or to perform transformation experiments on the plant itself. The entire paper needs to be rewritten with this point fully understood.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript successfully incorporates the reviewers' comments.