PONE-D-25-22089Transcriptome Analysis of Shade-Induced Growth and Photosynthetic Responses in Soybean CultivarsPLOS ONE

Dear Dr. Ren,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1:  The manuscript titled "Transcriptome Analysis of Shade-Induced Growth and

Photosynthetic Responses in Soybean Cultivars" is a comprehensive multi-level analysis

(morphological, physiological, transcriptomic) with strong data interpretation supported by

statistical testing. The manuscript have good potential for real-world application in breeding

shade-tolerant soybean cultivars. Following are the some major/minor corrections suggested

before the acceptance of the manuscript:

Major:

1. The P-Value used is 0.01 while no information about FDR. At least the p-value should be

0.05, which is a good statistical significance for DEGs. It is essential to apply multiple

testing correction, such as the Benjamini-Hochberg procedure, to control the false

discovery rate (FDR) during multiple comparisons.

2. The specific method used for DEG analysis is not mentioned.

Minor:

1. Too much general background on soybean and light effects, much of which is repeated in

later sections. Shorten and focus more on the research gap and novelty of this study

(Lines 39–80).

2. Inconsistent use of cultivar names throughout (Lines 20, 109, 200): "Guru" and "GURU"

are used interchangeably. Use consistent case and formatting for cultivar names.

3. The rationale is clear, but a formal hypothesis is not stated. Add a hypothesis or explicit

research objective at the end of the introduction (Lines 81–88).

4. Replicate numbers are not fully defined for RNA-seq experiments. Include biological

replicate numbers per treatment and confirm sequencing depth per sample (Lines

119–120, 209–215).

5. Lists of DEGs and comparisons (e.g., G70 vs. H70, GCK vs. HCK) are provided without

sufficient biological insight. Focus on top representative genes and discuss their

functional roles more deeply (Lines 217–238).

6. “Net photosynthetic rate (pr)” should be “(Pn)” for standardization. Correct abbreviation

to avoid misinterpretation (Line 180).

7. “One highly shade-tolerant” should be “One cultivar, Guru, which is highly shade-

tolerant” (Line 92), “do more photosynthesis” should be “perform more photosynthesis”

(Line 195).

8. Redundancy in the Discussion with Lines 296–338 and repeated in 334–344. Repetition

in describing chlorophyll content, photosynthesis, and light adaptation mechanisms.

Consolidate overlapping parts.

9. Incorrect figure reference in text. “Figure 3C” mentioned in the context of DEG

comparisons likely should be “Figure 4c”. Verify all figure citations match figure labels.

10. Improper listing (repeated GO terms without separators, e.g., “GO:0003712),

(GO:0004683)”). Ensure proper punctuation in gene ontology listings (Lines 259–260).

11. Consistency in writing numbers throughout the manuscript. Mix of numerical and word

formats (e.g., “three treatments” vs. “3 shade treatments”). Use consistent numerical

formatting.

Reviewer #2:  The manuscript entitled “Transcriptome Analysis of Shade-Induced Growth and Photosynthetic Responses in Soybean Cultivars” presents a well-designed and comprehensive study investigating the physiological, morphological, and transcriptomic responses of two contrasting soybean cultivars—Guru (shade-tolerant) and Heinong 53 (shade-sensitive)—under varying levels of shade (0%, 30%, and 70%). Through a combination of phenotypic analysis, chlorophyll and photosynthesis measurements, microscopic evaluation, and RNA sequencing, the authors demonstrate that Guru maintains higher photosynthetic efficiency, chlorophyll content, and a more stable resource allocation strategy under shade stress compared to Heinong 53, which exhibits excessive stem elongation and photosynthetic decline. The transcriptome analysis identified 2,596 differentially expressed genes (DEGs), with enriched functions related to shade avoidance, photosynthesis, and stress responses, including histone deacetylase and calcium-dependent kinase activity. Strong correlations between specific DEGs and physiological traits such as net photosynthetic rate and plant height were observed, supporting the molecular basis of phenotypic differences. This integrative work advances our understanding of shade tolerance mechanisms and provides valuable targets for breeding more resilient soybean cultivars. The manuscript is well-structured, methodologically sound, and the data are convincingly presented; I have no objections to its acceptance in its current form.

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Reviewer #1: No

Reviewer #2: No

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<p>Transcriptome Analysis of Shade-Induced Growth and Photosynthetic Responses in Soybean Cultivars

PONE-D-25-22089R1

Dear Dr. Ren,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Karthikeyan Adhimoolam

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: All review comments have been addressed by the authors with due care, improving the discourse and the research process.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

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