Responses of Different Japonica Rice Varieties to Cadmium Stress
Round 1
Reviewer 1 Report (Previous Reviewer 2)
Comments and Suggestions for AuthorsThe manuscript entitled “Study on Seedling Stage Response Characteristics and Cd Accumulation-Related Genes of Different Rice Varieties Under Cd Stress” focuses on investigating the Cd accumulation characteristics, physiological responses to stress, and relative expression levels of Cd-related transporter genes across different varieties. I have following comments and suggestions for improvement:
Introduction: The following corrections may be carried out for improvement of the manuscript,
Comment 1: Some of the grammatical and typographic errors in this section may be checked and corrected
Materials and methods: The following corrections may be carried out in the manuscript for its improvement,
Comment 2: Section 2.5, Line no. 180, include the unit of Cd content that was calculated
Comment 3: Pls check the chronology of the experiments. It is only after RNA extraction, qRT-PCR was performed, but was written vice-versa in the manuscript.
Comment 4: Section 2.7, provide standard references for all the derived parameters
Comment 5: Line 197, Ubiquitin gene was mentioned as reference, but, actin was mentioned in Table 1, pls clarify
Results: The following corrections may be carried out in the manuscript for its improvement,
Comment 6: The choice of Cd concentrations for hydroponic and soil experiments are reasonable, but justification for these levels relative to field contamination in Liaoning is missing
Comment 7: Hydroponic trials has three replicates, while soil culture uses six. justify
Comment 8: A figure showing plants/seedlings injury due to Cd stress in the evaluated varieties may be added.
Comment 9: The order of results may be modified/organized. Many results which were presented, did not have a detailed M&M. So it feels confusing and difficult to follow.
Discussion:
Comment 10: The authors cite previous studies but do not critically discuss contradictory findings. A deeper comparison would strengthen the argument that translocation is the dominant mechanism.
Comment 11: Connect physiological traits (growth, biomass, yield) with molecular findings to highlight breeding implications.
Abstract: Some of the sentences are not clear and scientifically unsound.
General comment: Grammatical and typographic errors may be checked and corrected. Some sentences should be modified for following scientific style of writing.
Comments on the Quality of English LanguageSome sentences are very verbose. Modify to maintain the scientific style of writing
Author Response
The manuscript entitled “Study on Seedling Stage Response Characteristics and Cd Accumulation-Related Genes of Different Rice Varieties Under Cd Stress” focuses on investigating the Cd accumulation characteristics, physiological responses to stress, and relative expression levels of Cd-related transporter genes across different varieties. I have following comments and suggestions for improvement:
Introduction: The following corrections may be carried out for improvement of the manuscript,
Comment 1: Some of the grammatical and typographic errors in this section may be checked and corrected.
Response 1: We thank the reviewer for the careful reading. We have thoroughly reexamined the entire Introduction section and corrected all identified grammatical and typographic errors.
Materials and methods: The following corrections may be carried out in the manuscript for its improvement,
Comment 2: Section 2.5, Line no. 180, include the unit of Cd content that was calculated
Response 2: We apologize for the omission. In the revised manuscript, we have added the unit “μg/g” at the indicated location and throughout Section 2.5.
Comment 3: Pls check the chronology of the experiments. It is only after RNA extraction, qRT-PCR was performed, but was written vice-versa in the manuscript.
Response 3: Thanks. We have carefully revised the relevant description in the manuscript to ensure the experimental sequence is accurate and consistent.
Comment 4: Section 2.7, provide standard references for all the derived parameters.
Response 4: We greatly appreciate the reviewer’s constructive suggestion. In Section 2.7, we have supplemented the corresponding standard references for all derived parameters to ensure the accuracy, authority and traceability of the parameter definitions and calculation methods.
Comment 5: Line 197, Ubiquitin gene was mentioned as reference, but, actin was mentioned in Table 1, pls clarify.
Response 5: We are grateful for this careful observation. We have verified the experimental methodology and uniformly revised both the text and Table 1 to use OsActin (LOC_Os03g50885) as the sole reference gene to eliminate ambiguity and ensure consistency.
Results: The following corrections may be carried out in the manuscript for its improvement,
Comment 6: The choice of Cd concentrations for hydroponic and soil experiments are reasonable, but justification for these levels relative to field contamination in Liaoning is missing.
Response 6: We thank the reviewer for the comment. We agree that the Cd concentrations used (1.0 mg·kg⁻¹ and 1.0 mg·L⁻¹) are higher than typical field levels in Liaoning. Our primary goal was mechanism discovery rather than field simulation. Higher concentrations were used to amplify genotypic differences in Cd transport efficiency, allowing clear detection of physiological and molecular responses. This approach is common in rice mechanism studies.
Comment 7: Hydroponic trials has three replicates, while soil culture uses six. justify.
Response 7: We thank the reviewer for the question. Soil culture used six replicates because of greater spatial heterogeneity of Cd in soil and longer growth duration (about 4 months), requiring higher statistical power. Hydroponic experiments used three replicates because the homogeneous solution conditions reduce individual variation, and this sample size is standard for early‑stage mechanistic studies in rice.
Comment 8: A figure showing plants/seedlings injury due to Cd stress in the evaluated varieties may be added.
Response 8: We thank the reviewer for the suggestion. In the revised manuscript, we have added a new figure (Figure S1, Supplementary Materials) showing representative seedlings of four rice varieties under control conditions and after 9 days of Cd stress.
Comment 9: The order of results may be modified/organized. Many results which were presented, did not have a detailed M&M. So it feels confusing and difficult to follow.
Response 9: We thank the reviewer for this constructive criticism, which has helped us significantly improve the clarity and logical flow of our manuscript. We have reorganized the Results section to follow a more logical, step‑by‑step sequence that mirrors the experimental design and research objectives. The revised structure is as follows:
“3.1. Growth and Biomass Responses to Cd Stress
3.1.1. Hydroponic Results at Seedling Stage
3.1.2. Soil Culture in Mature Stage Results
3.2. Cd Accumulation Characteristics and Varietal Differences
3.2.1. Tissue-Specific Cd Distribution Under Soil and Hydroponic Conditions
3.2.2. Enrichment and Transport Coefficients
3.2.2.1 Hydroponic Time-Course
3.2.2.2. Soil Culture
3.3. Antioxidant Defense System
3.4. Expression Profiles of Cd-Related Transporter Genes”
About enrichment of Materials and Methods. We have carefully reviewed all the results and added detailed descriptions of the methods where sufficient detail was previously lacking. The specific additions and revisions can be found on lines 232–234.
Discussion:
Comment 10: The authors cite previous studies but do not critically discuss contradictory findings. A deeper comparison would strengthen the argument that translocation is the dominant mechanism.
Response 10: We thank the reviewer for this insightful comment. We agree that simply citing previous studies without addressing contradictory findings weakens our argument. In the revised manuscript, we have substantially expanded the Discussion to critically compare our results with those from other studies, particularly those that have reported different rate‑limiting steps for Cd accumulation in rice.
Comment 11: Connect physiological traits (growth, biomass, yield) with molecular findings to highlight breeding implications.
Response 11: We have added a new paragraph in the Discussion (Section 4.3) that explicitly links physiological traits with molecular findings and highlights the breeding implications.
Abstract: Some of the sentences are not clear and scientifically unsound.
Response 12: We thank the reviewer for this critical comment. We have carefully reexamined the entire abstract and identified several sentences that were either ambiguous or scientifically imprecise. In the revised manuscript, we have substantially rewritten the abstract to improve clarity and scientific rigor. Below is a point‑by‑point explanation of the major changes.
Original sentence: “Cadmium (Cd) contamination in rice threatens food security.”. Revised sentence: “Cadmium (Cd) contamination in paddy soils threatens food security by accumulating in rice grains.”
Original sentence: “Cd content followed root > stem/leaf > grain.”. Revised sentence: “Cd distribution in rice plants followed the order: root > stem/leaf > grain.”
Original sentence: “Under Cd stress, low‑Cd varieties showed smaller MDA increases and stronger SOD/CAT induction.”. Revised sentence: “Low‑Cd varieties showed smaller MDA increases and significantly higher SOD and CAT activities under Cd stress.”
Original sentence: “The root to shoot translocation process and the opposite OsLCD expression pattern are key determinants differentiating low from high Cd japonica varieties.”. Revised sentence: “The root‑to‑shoot translocation process and the opposite OsLCD expression pattern are key determinants differentiating low‑ from high‑Cd japonica varieties.”
General comment: Grammatical and typographic errors may be checked and corrected. Some sentences should be modified for following scientific style of writing.
Response 13: We thank the reviewer for this careful reading and valuable suggestion. We have thoroughly reviewed the entire manuscript and corrected grammatical, typographical, and stylistic errors.
Reviewer 2 Report (New Reviewer)
Comments and Suggestions for AuthorsThe manuscript is well organized and provides useful physiological and molecular data on Cd accumulation in japonica rice, with good agreement between hydroponic and soil experiments. The discussion should be strengthened by better positioning the novelty relative to previous studies, clarifying the SN9903 pattern, and moderating the interpretation of OsLCD as an associated marker rather than a confirmed regulator. The limitations of using only four varieties, a relatively high hydroponic Cd level, and single time-point gene expression analysis should also be acknowledged more clearly.
Comments for author File: 
Comments.pdf
Author Response
Comment:The manuscript is well organized and provides useful physiological and molecular data on Cd accumulation in japonica rice, with good agreement between hydroponic and soil experiments. The discussion should be strengthened by better positioning the novelty relative to previous studies, clarifying the SN9903 pattern, and moderating the interpretation of OsLCD as an associated marker rather than a confirmed regulator. The limitations of using only four varieties, a relatively high hydroponic Cd level, and single time-point gene expression analysis should also be acknowledged more clearly.
Response :We thank the reviewer for the suggestion. In the revised manuscript, we have added a paragraph at the end of Discussion 4.3 that clearly acknowledges the limitations of using only four varieties, the relatively high hydroponic Cd level, and the single time‑point gene expression analysis. We also state that future extensive studies are needed to validate our findings.
Round 2
Reviewer 1 Report (Previous Reviewer 2)
Comments and Suggestions for AuthorsI have carefully reviewed the revised manuscript and the author’s responses to my comments. I am pleased to report that the authors have adequately addressed my suggestions and made the necessary revisions to improve the clarity and quality of the manuscript. I would suggest one more change to be made to improve the quality of the manuscript.
Comment 1: The phenotypic difference between low and high Cd accumulating genotypes for Cd content was not significant in roots. Then how do you justify using root tissues for expression studies.
Author Response
Comment 1: The phenotypic difference between low and high Cd accumulating genotypes for Cd content was not significant in roots. Then how do you justify using root tissues for expression studies.
Response 1: We thank the reviewer for this important comment. We would like to clarify that the samples for gene expression analysis were collected from shoot (leaf) tissues, not from roots. This was already specified in the Materials and Methods (Section 2.6, lines198) of our original manuscript.
To avoid any possible confusion and to make this point more explicit for readers, we have now added a clear statement at the beginning of Section 3.3. The added sentence reads:
“To investigate the molecular basis of varietal differences in Cd accumulation, we analyzed the expression of ten Cd-related transporter genes in stem and leaf under control and Cd stress conditions using qRT-PCR (Figure 6).”
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis manuscript addresses Cd contamination that threatens rice yield by investigating Cd accumulation, physiological responses, and the gene expression of Cd transporter genes in different rice varieties under hydroponic and soil Cd stress. The topic is important given its relevance to food safety and rice breeding.
However, there are some concerns that need to be further addressed.
Major comments:
- Novelty is limited and not clearly articulated. The study largely confirms well-known phenomena, including root > shoots > grains for Cd accumulation, low-Cd varieties have lower transport coefficients, antioxidant enzymes respond to Cd stress, known Cd transporters (NRAMPs, HMAs, LCD, etc) respond to Cd. Please clearly state: What is new compared with existing literature? What is the background of these four cultivars and why are they special? What unique insight does this study add beyond descriptive comparison?
- Why was 1 mg/L Cd²⁺ used for the treatment? Does it reflect realistic field exposure?
- Why is the Cd content in Figures 1 and 3 reported in mg/kg, while Cd accumulation in Figure 2 is reported in μg, and Cd accumulation in Figure 4a is reported in g. The units should be consistent.
- What is the statistical analysis method used in this study? ANOVA? Which post hoc test was used (LSD, Tukey’s HSD, Duncan)?
Minor comments:
- All abbreviations should be defined at first appearance (e.g. ICP-MS, ICP-OES)
- Line 37, extra empty space after [1].
- Lines 60-67, sentences in these lines are overly long and lack appropriate punctuation and clear sentence boundaries.
- Lines 103-104, how was it determined that “YF47 and SN9903 are low Cd-accumulating varieties, while QTXT and TJ are high Cd-accumulating varieties.”?
- Were the primers used in this study (listed in Table S1) designed by the authors or adopted from previously published studies? If they are adopted from previous published studies, please provide the corresponding references.
- There are several typos in the manuscript. In line 42, “it ios” should be corrected to “it is? In the table headers of Table 2, “plant heigh (cm)” should be corrected to “plant height (cm)”, and “root weigh(mg)” should be corrected to “root weight (mg)”. In table headers of Table 3, “Relative root weigh (%)” should be corrected to “Relative root weight (%)”.
- The reference formate should be standardized.
Reviewer 2 Report
Comments and Suggestions for Authors-Attached-
Comments for author File: Comments.pdf
