Review Reports - Construction of a Yeast Two-Hybrid cDNA Library from Soybean Seeds at Different Developmental Stages and Screening of SW16.1 Interacting Proteins

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study focuses on the screening of interacting proteins of soybean GmSW16.1 and their potential roles in seed development. The authors employed a yeast two-hybrid library screening approach, validation of candidate interacting proteins, analysis of PolyQ polymorphisms, and Arabidopsis overexpression assays to provide preliminary insights into the potential molecular mechanism by which GmSW16.1 may participate in seed development. Overall, the experimental design is relatively complete, and the results provide useful information. However, several aspects of the manuscript, including the use of certain technical terms, the rigor of experimental result interpretation, the association between candidate interacting proteins and seed development, and English language and formatting, could be further improved. I recommend that the authors address the following points.

The concepts of “interaction strength” and “binding affinity” should be distinguished more precisely.
Some results in the manuscript are based on the yeast two-hybrid system and α-galactosidase reporter activity, which are used to compare the relative strength of interactions between different proteins. However, the manuscript uses terms such as “binding affinity” to describe these differences. Y2H reporter activity primarily reflects the strength of the interaction-associated reporter signal under the specific experimental conditions and cannot be directly equated with biochemical binding affinity. Therefore, the authors are encouraged to replace such terminology with more appropriate terms, such as “interaction strength,” “interaction intensity,” or “interaction-associated reporter activity,” unless direct biochemical binding assays are provided. This revision would make the interpretation of the interaction results more scientifically rigorous.
The expression patterns of candidate interacting proteins could be further analyzed to support their potential association with seed development.
Since the Y2H library used in this study was constructed from soybean seeds at different developmental stages, the authors could consider providing expression information for the major candidate interacting proteins. For example, qRT-PCR analysis of key candidates such as GmMT4 and Glyma.08G116300 at several representative seed developmental stages would be helpful. Such analysis could provide additional information on the expression patterns of these candidates during seed development and further strengthen the biological relevance of the Y2H screening results.
The association between Glyma.08G116300 and seed developmental phenotypes should be further strengthened.
At present, the Arabidopsis overexpression experiments mainly evaluate the potential function of Glyma.08G116300 based on traits such as 1000-seed weight. If feasible, the authors could provide additional and relatively straightforward seed-related phenotypic measurements, together with representative photographs, such as seed length, seed width, or seed area. These measurements would provide a more comprehensive characterization of the seed phenotype in the overexpression lines. In addition, if the relevant materials and experimental conditions are available, expression analysis of Glyma.08G116300 in soybean seeds at different developmental stages could also be considered to further support its potential association with soybean seed development.
The English language, grammar, and formatting should be further checked and standardized throughout the manuscript.
The manuscript is generally well written in English, with a clear logical structure and relatively rigorous presentation of the experimental results, and overall follows the conventions of plant science research articles. However, there are still some minor issues related to language and formatting, such as missing hyphens in compound terms, inconsistent use of punctuation and spacing, and occasional redundant wording. The authors are encouraged to carefully proofread the entire manuscript and further standardize technical terminology, punctuation, and formatting to improve the overall clarity, consistency, and readability of the manuscript.

Author Response

Dear Reviewer,

We sincerely thank the reviewer for you careful and constructive evaluation of our manuscript. The comments were highly valuable and have helped us substantially improve the clarity, rigor, and presentation of this work. We have revised the manuscript point by point in accordance with all the comments. In the revised manuscript, newly added or substantially modified text is highlighted in red so that the changes can be readily identified. Below, we respond to each comment of the reviewer in a point-by-point manner. Reviewer comments are reproduced in italics, and the corresponding author responses follow.

Response to Reviewer 1

We thank Reviewer 1 for the positive assessment of our experimental design and for the constructive suggestions regarding terminology, expression analyses, phenotypic characterization, and language. We have addressed all the points raised, as detailed below.

Comment: The concepts of “interaction strength” and “binding affinity” should be distinguished more precisely. Some results in the manuscript are based on the yeast two-hybrid system and α-galactosidase reporter activity, which are used to compare the relative strength of interactions between different proteins. However, the manuscript uses terms such as “binding affinity” to describe these differences. Y2H reporter activity primarily reflects the strength of the interaction-associated reporter signal under the specific experimental conditions and cannot be directly equated with biochemical binding affinity. Therefore, the authors are encouraged to replace such terminology with more appropriate terms, such as “interaction strength,” “interaction intensity,” or “interaction-associated reporter activity,” unless direct biochemical binding assays are provided.

Response: We agree with the reviewer. Throughout the manuscript, we describe the Y2H and quantitative α-galactosidase results with the terms “interaction strength” and “interaction-associated reporter activity,” and we do not equate these measurements with a directly determined biochemical binding affinity. This distinction is explicitly stated in Section 4.4 of the revised manuscript: “the α-galactosidase reporter activity measured here reflects the interaction-associated reporter signal under the specific yeast assay conditions, it is used comparatively as a proxy for interaction strength between alleles, and is not equivalent to a directly determined biochemical binding affinity, which would require in vitro binding assays with purified proteins.”

We have also carefully proofread the entire manuscript to ensure that these terms are used consistently and that no Y2H-based result is described as a directly measured “binding affinity.”

Comment: The expression patterns of candidate interacting proteins could be further analyzed to support their potential association with seed development. Since the Y2H library used in this study was constructed from soybean seeds at different developmental stages, the authors could consider providing expression information for the major candidate interacting proteins. For example, qRT-PCR analysis of key candidates such as GmMT4 and Glyma.08G116300 at several representative seed developmental stages would be helpful.

Response: We thank the reviewer for this valuable suggestion. In the revised manuscript, we provide qRT-PCR analysis of Glyma.08G116300 transcript abundance at multiple representative seed developmental stages (21, 28, 35, and 42 DAF) in both the chromosome segment substitution line CSSL3068 and its cultivated parent Nannong 1138-2 (revised Section 3.5, Figure 6C). We have rewritten this section for clarity, and we have added a concise summary to the Abstract: “its transcript abundance differed dynamically between the chromosome segment substitution line CSSL3068 and its cultivated parent Nannong 1138-2 across developmental stages.”

Revised text: “We further analyzed the relative transcript abundance of Glyma.08G116300 in the two parents of the chromosome segment substitution line at different seed developmental stages (Figure 6C). Transcript levels did not differ significantly between the two parents at the early stage (21 DAF). At the mid-stage (28 DAF), expression in CSSL3068 was significantly higher than that in the cultivated soybean Nannong 1138-2 (NN1138-2). By contrast, expression in NN1138-2 was significantly higher than in CSSL3068 at later stages (35 and 42 DAF). These results suggest that Glyma.08G116300 may be involved in soybean seed development.”

Regarding GmMT4, its seed-development-stage expression and its positive correlation with soybean seed weight have been previously characterized [17]; this is cited in Sections 3.5 and 4.3 to support the seed-associated biological relevance of the Y2H screening.

Comment: The association between Glyma.08G116300 and seed developmental phenotypes should be further strengthened. At present, the Arabidopsis overexpression experiments mainly evaluate the potential function of Glyma.08G116300 based on traits such as 1000-seed weight. If feasible, the authors could provide additional and relatively straightforward seed-related phenotypic measurements, together with representative photographs, such as seed length, seed width, or seed area. In addition, if the relevant materials and experimental conditions are available, expression analysis of Glyma.08G116300 in soybean seeds at different developmental stages could also be considered to further support its potential association with soybean seed development.

Response: We appreciate the reviewer’s constructive suggestion. In the revised manuscript we clearly document the seed-size phenotyping. The measurement procedure is now described in Section 2.7: “The length and width of fifteen seeds per line were measured using a stereomicroscope.” Representative photographs of mature seeds of the wild-type (WT) and the three overexpression lines (OE-1, OE-7, OE-11) are presented in Figure 6F, and the seed length and width data are shown in Figure 6G, H. The results are now explicitly stated in the revised Section 3.5: “The seed length and width of the transgenic lines were significantly greater than those of the WT (Figure 6F–H).”

Accordingly, the Abstract now states: “Heterologous overexpression of this gene in Arabidopsis thaliana significantly increased the 1000-seed weight (by 12%–15%) and increased seed length and width.” In addition, as described in the response to Comment 1.2 above, qRT-PCR expression analysis of Glyma.08G116300 in soybean seeds at several developmental stages (21–42 DAF) is provided (Figure 6C), supporting its potential association with soybean seed development.

Comment: The English language, grammar, and formatting should be further checked and standardized throughout the manuscript. The manuscript is generally well written in English ... However, there are still some minor issues related to language and formatting, such as missing hyphens in compound terms, inconsistent use of punctuation and spacing, and occasional redundant wording. The authors are encouraged to carefully proofread the entire manuscript and further standardize technical terminology, punctuation, and formatting.

Response: We thank the reviewer for the careful reading. The entire manuscript has been thoroughly proofread and revised for language, grammar, and formatting. In particular, we (i) corrected hyphenation in compound terms (e.g., “wild-type” and “ribosomal-ubiquitin”); (ii) standardized punctuation, spacing, and technical terminology throughout; and (iii) removed redundant or awkward wording, for example the duplicated phrase in the original Figure 6C legend and the grammatically incorrect sentence in Section 2.7.

We also corrected the inconsistent statistical wording in the Figure 6 legend and the formatting of reference entries and realigned the reference list with the citation order. The revised manuscript has been re-checked to ensure consistency and readability. In the revised manuscript, newly added or substantially modified text is highlighted in red.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Authors described the construction of a seed-development-specific Y2H cDNA library from soybean cultivar Nannong 1138-2, a screen for GmSW16.1-interacting proteins, and multi-platform validation of candidates, focusing on GmMT4 and Glyma.08G116300. The study acquired a valuable community resource and a dual-layer GmSW16.1 regulatory module, with generally clear writing and proper discussion of limitations. It is good for soybean and seed biology research but minor-to-moderate revisions is required prior to publication.

First, the core mechanistic novelty might be little concerned. GmSW16.1-mediated transcriptional regulation of GmMT4 has been previously published. The novel findings—physical protein interaction and PolyQ-length-dependent binding affinity modulation—are valid but might be overstated. The “dual-layer module” should be explicitly framed as a working hypothesis, with proper presentation in the abstract and conclusions.

Second, candidate validation might be incomplete and preliminary. Of 11 identified interactors, five were further studied, and just two were confirmed via BiFC. The functional claim for Glyma.08G116300 as a seed weight positive regulator is still unsubstantiated, relying solely on Arabidopsis heterologous overexpression without loss-of-function, soybean transformation, or rescue assays, et al.

Additionally, PolyQ-based affinity tuning interpretation is speculative. Protein expression verification and in planta BiFC signal quantification are required to support claims. Critical soybean library quality metrics (recombinant clone ratio, average insert size, total independent clones) are also missing

There are inconsistent data in Figure 5, unclear gene numbering, and insufficiently annotated Phytozome expression data. The RNA pooling strategy’s limitation of under-recovering narrow-window expressed genes should be noted. Statistical methods need optimization: ANOVA with post-hoc tests should replace single t-tests for multiple comparisons. Minor typos, formatting errors, and misaligned references need to be fixed.

Overall, This work provides a reusable seed development cDNA library with rigorous experimental workflows. This manuscript merits publication after targeted revisions

Author Response

Dear Reviewer,

Response to Reviewer 2

We sincerely thank Reviewer 2 for recognizing the value of the seed-development Y2H cDNA library and the rigorous experimental workflow. We have carefully considered all the comments and revised the manuscript accordingly, as detailed below.

Comment: First, the core mechanistic novelty might be little concerned. GmSW16.1-mediated transcriptional regulation of GmMT4 has been previously published. The novel findings—physical protein interaction and PolyQ-length-dependent binding affinity modulation—are valid but might be overstated. The “dual-layer module” should be explicitly framed as a working hypothesis, with proper presentation in the abstract and conclusions.

Response: We agree with the reviewer. The dual-layer GmSW16.1–GmMT4 regulatory module is explicitly presented as a “testable working hypothesis” in the Abstract, in Section 4.3, and in the Conclusions, rather than as an established mechanism. We have also moderated the wording of our claims throughout the manuscript so that the proposed module and its functional implications are clearly framed as hypotheses to be tested in future work. For example, the revised Section 3.5 states: “These results indicate that Glyma.08G116300 positively influences seed development in a heterologous background.” This revised wording avoids overstating the novelty and the functional conclusions.

Comment: Second, candidate validation might be incomplete and preliminary. Of 11 identified interactors, five were further studied, and just two were confirmed via BiFC. The functional claim for Glyma.08G116300 as a seed weight positive regulator is still unsubstantiated, relying solely on Arabidopsis heterologous overexpression without loss-of-function, soybean transformation, or rescue assays, et al.

Response: We fully acknowledge this critical comment. In the revised manuscript, we have deliberately softened the functional claims for Glyma.08G116300. Section 3.5 now concludes that the gene “positively influences seed development in a heterologous background,” rather than being established as a soybean seed-weight regulator. In Section 4.5, we explicitly state the limitation of the current evidence and the need for loss-of-function, stable soybean transformation, and rescue experiments: “However, the current phenotypic evidence comes exclusively from Arabidopsis heterologous overexpression. We lack loss-of-function knockout and stable soybean overexpression data to confirm its endogenous function in soybean. Therefore, we cannot definitively conclude that Glyma.08G116300 is an endogenous positive regulator of soybean seed-weight.”

Revised text: We also added the following paragraph to Section 4.5 to integrate the CSSL3068 expression data and further temper the interpretation: “The developmental expression divergence of Glyma.08G116300 between CSSL3068 and Nannong 1138-2, higher in CSSL3068 at 28 DAF but higher in Nannong 1138-2 at 35 and 42 DAF, provides additional evidence linking this gene to seed developmental progression. ... Nevertheless, the stage-dependent direction of this expression difference warrants cautious interpretation, and the endogenous function of Glyma.08G116300 in soybean seed-weight control still requires loss-of-function and stable transgenic validation.”

The corresponding claims in the Abstract and Conclusions have likewise been reworded to reflect the hypothesis-driven and preliminary nature of the functional evidence.

Comment: Additionally, PolyQ-based affinity tuning interpretation is speculative. Protein expression verification and in planta BiFC signal quantification are required to support claims. Critical soybean library quality metrics (recombinant clone ratio, average insert size, total independent clones) are also missing.

Response: We thank the reviewer for these points. (i) Regarding the PolyQ-based affinity-tuning interpretation, this model is explicitly presented as a hypothesis in Section 4.4, and we state that direct experimental validation using recombinant GmSW16.1 and GsSW16.1 proteins (including in vitro binding assays) is required. The conformational interpretation is clearly marked as inferred from published structural studies. We acknowledge that protein-expression verification and quantitative in planta BiFC signal measurement would further strengthen the analysis, and we have noted these as important future work. (ii) Regarding the soybean library quality metrics, these are already provided in Section 2.2 and Section 3.1: the library titre (2 × 10⁷ cfu/mL, exceeding the 1 × 10⁷ cfu/mL benchmark); the recombination rate, assessed by PCR amplification of prey inserts in 48 randomly picked colonies, with nearly all colonies carrying inserts; the insert-size distribution, broadly ranging from approximately 500 to 2500 bp among the tested clones and consistent with the ds-cDNA smear of approximately 300 to 6000 bp; and the total number of independent recombinant clones, calculated as the library titre multiplied by the total volume of the stored aliquot.

Comment: There are inconsistent data in Figure 5, unclear gene numbering, and insufficiently annotated Phytozome expression data. The RNA pooling strategy’s limitation of under-recovering narrow-window expressed genes should be noted. Statistical methods need optimization: ANOVA with post-hoc tests should replace single t-tests for multiple comparisons. Minor typos, formatting errors, and misaligned references need to be fixed.

Response: We thank the reviewer for these detailed and helpful points. (1) The Figure 5 legend has been clarified to describe the gene-structure schematic, the functional-domain positions, the hydropathy profile, and the homology-derived three-dimensional model, ensuring consistency with the data presented. (2) The Phytozome expression data are now clearly described in Section 2.8 (data source, assembly, and access date) and annotated in Figure 6B with FPKM values. (3) The limitation of the multi-stage RNA pooling strategy in under-recovering genes expressed in a narrow developmental window is explicitly noted in Section 4.1: “pooling may dilute low abundance transcripts that are restricted to a narrow developmental window.” (4) All multiple comparisons have been analyzed by one-way ANOVA followed by Tukey’s post-hoc test (Section 2.5), and the Figure 6 legend now states “** p < 0.01 by one-way ANOVA with Tukey’s post-hoc test” (replacing the previous wording). (5) Typos, formatting errors, and reference misalignment have been corrected, including the format of [36] reference entry and the reordering of the reference list to match the citation order in the text.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by Xingchen Liu et al describes the construction of a yeast two-hybrid (Y2H) cDNA library from soybean seeds collected at five developmental stages and its application to identify proteins interacting with SW16.1, a gene associated with seed weight. The study combines library screening, point-to-point Y2H assays, quantitative α-galactosidase measurements, bimolecular fluorescence complementation (BiFC), and heterologous overexpression in Arabidopsis. The identification of GmMT4 and Glyma.08G116300 as SW16.1 interaction partners is potentially interesting, particularly in view of the comparison between cultivated and wild soybean alleles. The experimental approach adopted by the Authors is appropriate, and the results are generally well supported by the experiments performed. This Reviewer appreciate the comprehensive experimental work and the combination of complementary techniques used to investigate the protein–protein interactions and the potential role of Glyma.08G116300 in seed-weight regulation. However, before the manuscript is accepted for publication, the Authors should provide more details about the procedure used for weight seed experiment: sampling procedure about collection techniques, number of seed collected per plant and for independent group, etc.

Comments on the Quality of English Language

English should be carefully revised by a professional language editor.

Author Response

Dear Reviewer,

Response to Reviewer 3

We thank Reviewer 3 for the positive assessment of the experimental approach and for the appreciation of the complementary techniques used in this study. We have addressed the requested clarification of the seed-weight experiment procedure as detailed below.

Comment: However, before the manuscript is accepted for publication, the Authors should provide more details about the procedure used for weight seed experiment: sampling procedure about collection techniques, number of seed collected per plant and for independent group, etc.

Response: We thank the reviewer for this request. In the revised Section 2.7, we now provide the detailed sampling and measurement procedures for the 1000-seed-weight experiment: “For 1000-seed weight measurement, wild-type and transgenic plants were grown simultaneously under identical growth-chamber conditions (16 h light/8 h dark, 22 °C, 60% relative humidity). Mature siliques were bulk-harvested separately from at least 3 plants per line, and fully filled seeds were collected, air-dried at room temperature for two weeks, and stored under dry conditions. For each biological replicate, 1000 fully developed seeds were counted manually and weighed using an analytical balance (readability 0.1 mg). Three independent biological replicates were measured per line.”

In addition, we clarify that for seed-size phenotyping, the length and width of fifteen seeds per line were measured using a stereomicroscope, as described in Section 2.7 and shown in Figure 6G, H.

We hope that the revised manuscript now meets the standards of Plants. We are grateful for the reviewers’ time and for their detailed and constructive comments, which have considerably improved the manuscript. We would be happy to make any further revisions if required.

Author Response File: Author Response.docx