Review Reports

Response to Reviewer 1 Comments

1. Summary

We sincerely appreciate the time and effort you have dedicated to reviewing this manuscript. Detailed responses were provided below, and corresponding revisions/corrections were highlighted or marked via track changes in the resubmitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

According to the corresponding comments, we have detailed revised this section in the point-by-point response letter as follows.

Is the research design appropriate?

Yes

Are the methods adequately described?

Can be improved

According to the corresponding comments, we have detailed revised this section in the point-by-point response letter as follows.

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Can be improved

According to the corresponding comments, we have detailed revised this section in the point-by-point response letter as follows.

Are all figures and tables clear and well-presented?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The "Introduction" section should be improved by adding bibliographic references that address drought stress in different plant species and the genes responsible for wax synthesis. It would be good to also mention the composition of the wax, the thickness of the wax layer depending on the species, in the context of the specific geografical area.

Response 1: Thanks for your suggestion. We have enhanced the 'Introduction' by adding relevant references on drought stress, wax synthesis genes, and species-specific wax composition/thickness.

In lines 30–31: “Drought is one of the most severe disasters worldwide, posing significant challenges to plant growth and survival [1,2].” was revised as “Drought is one of the primary abiotic stresses limiting plant growth and crop yield [1,2]. Analyzing the mechanism of drought's impact on plant growth [3], clarifying the molecular regulatory network of plants in response to drought stress [4], and identifying key drought-resistant gene [5] can not only enrich and improve the theoretical framework of plant stress biology, but also provide important theoretical support and gene reserves for drought-resistant molecular design breeding of crops.”

In line 33: “including key drought-resistant traits such as cuticular wax synthesis [8,9], stomatal development and movement [10,11], root growth [12–14], and reproductive development [15,16].” was added.

In line 36: “cuticular wax was mainly composed of very-long-chain fatty acids and their derivatives [19,20]. It effectively” was added.

In References section,

“3. Verma, K.K.; Song, X.P.; Kumari, A.; Jagadesh, M.; Singh, S.K.; Bhatt, R.; Singh, M.; Seth, C.S.; Li, Y.R. Climate change adaptation: Challenges for agricultural sustainability. Plant Cell Environ. 2025, 48, 2522–2533.

4. Shah, W.U.H.; Lu, Y.; Liu, J.; Rehman, A.; Yasmeen, R. The impact of climate change and production technology heterogeneity on China's agricultural total factor productivity and production efficiency. Sci Total Environ. 2024, 907, 168027.

5. Chen, K.; Gao, J.; Sun, S.; Zhang, Z.; Yu, B.; Li, J.; Xie, C.; Li, G.; Wang, P.; Song, C.P.; et al. BONZAI proteins control global osmotic stress responses in plants. Curr Biol. 2020, 30, 4815–4825.

8. Lian, X.Y.; Gao, H.N.; Jiang, H.; Liu, C.; Li, Y.Y. MdKCS2 increased plant drought resistance by regulating wax biosynthesis. Plant Cell Rep. 2021, 40, 2357–2368.

9. Urano, K.; Oshima, Y.; Ishikawa, T.; Kajino, T.; Sakamoto, S.; Sato, M.; Toyooka, K.; Fujita, M.; Kawai-Yamada, M.; Taji, T.; et al. Arabidopsis DREB26/ERF12 and its close relatives regulate cuticular wax biosynthesis under drought stress condition. Plant J. 2024, 120, 2057–2075.

10. Liu, C.; Sack, L.; Li, Y.; Zhang, J.; Yu, K.; Zhang, Q.; He, N.; Yu, G. Relationships of stomatal morphology to the environment across plant communities. Nat Commun. 2023, 14, 6629.

11. Liu, L.; Ashraf, M.A.; Morrow, T.; Facette, M. Stomatal closure in maize is mediated by subsidiary cells and the PAN2 receptor. New Phytol. 2024, 241, 1130–1143.

12. Sun, X.; Xiong, H.; Jiang, C.; Zhang, D.; Yang, Z.; Huang, Y.; Zhu, W.; Ma, S.; Duan, J.; Wang, X.; et al. Natural variation of DROT1 confers drought adaptation in upland rice. Nat Commun. 2022, 13, 4265.

13. Han, S.; Wang, Y.; Li, Y.; Zhu, R.; Gu, Y.; Li, J.; Guo, H.; Ye, W.; Nabi, H.G.; Yang, T.; et al. The OsNAC41-RoLe1-OsAGAP module promotes root development and drought resistance in upland rice. Mol Plant. 2024, 17, 1573–1593.

14. Zhang, X.; Mi, Y.; Mao, H.; Liu, S.; Chen, L.; Qin, F. Genetic variation in ZmTIP1 contributes to root hair elongation and drought tolerance in maize. Plant Biotechnol J. 2020, 18, 1271–1283.

15. Ying, S.; Scheible, W.R.; Lundquist, P.K. A stress-inducible protein regulates drought tolerance and flowering time in Brachypodium and Arabidopsis. Plant Physiol. 2023, 191, 643–659.

16. Shavrukov, Y. Pathway to the molecular origins of drought escape and early flowering illuminated via the phosphorylation of SnRK2-Substrate 1 in Arabidopsis. Plant Cell Physiol. 2024, 65, 179–180.

19. Bernard, A.; Domergue, F.; Pascal, S.; Jetter, R.; Renne, C.; Faure, J.D.; Haslam, R.P.; Napier, J.A.; Lessire, R.; Joubès, J. Reconstitution of plant alkane biosynthesis in yeast demonstrates that Arabidopsis ECERIFERUM1 and ECERIFERUM3 are core components of a very-long-chain alkane synthesis complex. Plant Cell. 2012, 24, 3106–3118

20. Rowland, O.; Zheng, H.; Hepworth, S.R.; Lam, P.; Jetter, R.; Kunst, L. CER4 encodes an alcohol-forming fatty acyl-coenzyme A reductase involved in cuticular wax production in Arabidopsis. Plant Physiol. 2006, 142, 866–877.” were added.

Comments 2: Is correct 25 ℃, not 25℃. You should mention the trade name of the climate chamber.

Response 2: Thank you for your reminder, we have made corresponding revisions based on your suggestions.

In line 72: “25℃” was corrected as “25 ℃”.

In line 71: trade name of the climate chamber “Hangzhou Zhizai Biotechnology CO., LTD.” was added.

Comments 3: In line 78 it is mentioned by "physiological index determination" and in this regard, they have determined: SOD, NBT, POD, MDA, TBA, Pro. In that case, "physiological index" should be replaced with "physiological indicators" as you mentioned in line 81.

Response 3: Based on your suggestion, in line 81: the "physiological index" was replaced with "physiological indicators".

Comments 4: Althought you mention abbreviations in "Material and Methods", you should add a list of abbreviations after "Conclusions", to facilitate understanding of this study.

Response 4: As recommended, a list of abbreviations was supplemented after Conclusions to facilitate study comprehension, as follow.

Comments 5: In line 362, Arabidopsis thaliana should be written in italics.

Response 5: Based on your suggestion, in line 362: the “Arabidopsis thaliana” was corrected as italics.

Comments 6: The conclusions could be enriched according to the multitude of data presented.

Response 6: Thank you for your comment. We have enriched the Conclusions section by integrating key data from the study.

In lines 400–401: “This study, a pair of Chinese cabbage NILs was used, has for the first time revealed the phenotypic and physiological response patterns under drought stress.” was revised as “In this study, a pair of Chinese cabbage waxy NILs was used for the first time to reveal the dynamic patterns of plant phenotype, physiology, and gene expression under drought stress. Phenotypic and physiological data demonstrated that waxy ‌T065-1‌ exhibited significantly higher antioxidant capacity and stress adaptability compared to the non-waxy ‌T065-2‌ under drought stress.”

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response: Thank you for acknowledging that the English language quality of this manuscript meets the requirements. We will maintain rigorous language expression in subsequent revisions to uphold this quality standard.

5. Additional clarifications

Response: Additional clarifications on details were provided in the revised manuscript.

Response to Reviewer 2 Comments

1. Summary

Thank you for your insightful feedback. We appreciate your valuable comments and will carefully address the gaps to improve the manuscript.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Can be improved

According to the corresponding comments, we have detailed revised this section in the point-by-point response letter as follows.

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

Are all figures and tables clear and well-presented?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The abstract is very informative and properly structured; no major revisions are needed. However, the authors may consider partially modifying the final sentence to emphasize the impact or biotechnological application of their findings, for instance, how these results could contribute to the breeding of drought-resistant crops.

Response 1: Thanks, we revised the final sentence to highlight the biotechnological impact and crop breeding potential.

In lines 24–26: “These findings provide a crucial theoretical foundation for the development of drought-resistant molecular markers and molecular target editing in Chinese cabbage.” was revised as “These findings provide a crucial theoretical foundation for exploring drought-resistant molecular markers and editing targets in Chinese cabbage, significantly accelerating the breeding of superior drought-resistant varieties.”

Comments 2: Introduction section is engaging and effectively contextualizes the drought problem and the role of cuticular wax in stress tolerance, supported by appropriate references and a clearly stated objective.

Nonetheless, while solid, it extends excessively into examples from other crops (rice, apple, wheat). Although this is not inherently problematic, it would be valuable to integrate how these findings justify focusing on Chinese cabbage. The authors could link physiology to genomics to demonstrate conceptual depth, rather than listing previous studies.

A minor correction: in line 41, at its first mention, please use “Wilted Dwarf and Lethal (WDL)”. Additionally, in line 45, periods are missing after MdMIEL1.

Response 2: Thank you for your insightful comments. We have integrated physiology with  genomics to enhance the biological significance of this study.

In lines 30–31: “Drought is one of the most severe disasters worldwide, posing significant challenges to plant growth and survival [1,2].” was revised as “Drought is one of the primary abiotic stresses limiting plant growth and crop yield [1,2]. Analyzing the mechanism of drought's impact on plant growth [3], clarifying the molecular regulatory network of plants in response to drought stress [4], and identifying key drought-resistant gene [5] can not only enrich and improve the theoretical framework of plant stress biology, but also provide important theoretical support and gene reserves for drought-resistant molecular design breeding of crops.”

In line 33: “including key drought-resistant traits such as cuticular wax synthesis [8,9], stomatal development and movement [10,11], root growth [12–14], and reproductive development [15,16].” was added.

In line 36: “cuticular wax was mainly composed of very-long-chain fatty acids and their derivatives [19,20]. It effectively” was added.

In line 41: “WDL” was revised as “Wilted Dwarf and Lethal 1 (WDL1)”. In line 45: the missing space after the “MdMIEL1” has been added.

In References section,

“3. Verma, K.K.; Song, X.P.; Kumari, A.; Jagadesh, M.; Singh, S.K.; Bhatt, R.; Singh, M.; Seth, C.S.; Li, Y.R. Climate change adaptation: Challenges for agricultural sustainability. Plant Cell Environ. 2025, 48, 2522–2533.

4. Shah, W.U.H.; Lu, Y.; Liu, J.; Rehman, A.; Yasmeen, R. The impact of climate change and production technology heterogeneity on China's agricultural total factor productivity and production efficiency. Sci Total Environ. 2024, 907, 168027.

5. Chen, K.; Gao, J.; Sun, S.; Zhang, Z.; Yu, B.; Li, J.; Xie, C.; Li, G.; Wang, P.; Song, C.P.; et al. BONZAI proteins control global osmotic stress responses in plants. Curr Biol. 2020, 30, 4815–4825.

8. Lian, X.Y.; Gao, H.N.; Jiang, H.; Liu, C.; Li, Y.Y. MdKCS2 increased plant drought resistance by regulating wax biosynthesis. Plant Cell Rep. 2021, 40, 2357–2368.

9. Urano, K.; Oshima, Y.; Ishikawa, T.; Kajino, T.; Sakamoto, S.; Sato, M.; Toyooka, K.; Fujita, M.; Kawai-Yamada, M.; Taji, T.; et al. Arabidopsis DREB26/ERF12 and its close relatives regulate cuticular wax biosynthesis under drought stress condition. Plant J. 2024, 120, 2057–2075.

10. Liu, C.; Sack, L.; Li, Y.; Zhang, J.; Yu, K.; Zhang, Q.; He, N.; Yu, G. Relationships of stomatal morphology to the environment across plant communities. Nat Commun. 2023, 14, 6629.

11. Liu, L.; Ashraf, M.A.; Morrow, T.; Facette, M. Stomatal closure in maize is mediated by subsidiary cells and the PAN2 receptor. New Phytol. 2024, 241, 1130–1143.

12. Sun, X.; Xiong, H.; Jiang, C.; Zhang, D.; Yang, Z.; Huang, Y.; Zhu, W.; Ma, S.; Duan, J.; Wang, X.; et al. Natural variation of DROT1 confers drought adaptation in upland rice. Nat Commun. 2022, 13, 4265.

13. Han, S.; Wang, Y.; Li, Y.; Zhu, R.; Gu, Y.; Li, J.; Guo, H.; Ye, W.; Nabi, H.G.; Yang, T.; et al. The OsNAC41-RoLe1-OsAGAP module promotes root development and drought resistance in upland rice. Mol Plant. 2024, 17, 1573–1593.

14. Zhang, X.; Mi, Y.; Mao, H.; Liu, S.; Chen, L.; Qin, F. Genetic variation in ZmTIP1 contributes to root hair elongation and drought tolerance in maize. Plant Biotechnol J. 2020, 18, 1271–1283.

15. Ying, S.; Scheible, W.R.; Lundquist, P.K. A stress-inducible protein regulates drought tolerance and flowering time in Brachypodium and Arabidopsis. Plant Physiol. 2023, 191, 643–659.

16. Shavrukov, Y. Pathway to the molecular origins of drought escape and early flowering illuminated via the phosphorylation of SnRK2-Substrate 1 in Arabidopsis. Plant Cell Physiol. 2024, 65, 179–180.

19. Bernard, A.; Domergue, F.; Pascal, S.; Jetter, R.; Renne, C.; Faure, J.D.; Haslam, R.P.; Napier, J.A.; Lessire, R.; Joubès, J. Reconstitution of plant alkane biosynthesis in yeast demonstrates that Arabidopsis ECERIFERUM1 and ECERIFERUM3 are core components of a very-long-chain alkane synthesis complex. Plant Cell. 2012, 24, 3106–3118

20. Rowland, O.; Zheng, H.; Hepworth, S.R.; Lam, P.; Jetter, R.; Kunst, L. CER4 encodes an alcohol-forming fatty acyl-coenzyme A reductase involved in cuticular wax production in Arabidopsis. Plant Physiol. 2006, 142, 866–877.” were added.

Additionally, other minor details have been revised in the revised manuscript.

Comments 3: Materials and Methods，Overall, this section is coherent and well-organized. However, several important details should be addressed: 

2.1. Plant Materials and Drought Treatment

(1) The volume of the Hoagland solution and the frequency of renewal or aeration are not specified, which affects reproducibility.

(2) The number of plants per replicate (“three biological replicates”) is not clarified, nor are the selection criteria for sampled leaves.

(3) There is no information on how uniformity in plant age or size was controlled between treatments.

(4) The 10% PEG level is mentioned without justification or citation; a reference supporting this concentration for Brassica or a similar species should be provided.

Response 3: We appreciate your feedback on Materials and Methods, in this study, when the seedlings reached the three-leaf-one-heart stage, all were transferred into 100× Hoagland nutrient solution for hydroponic culture. The control group continued to grow in 100× Hoagland solution for 24 hours, while the treatment group was successively subjected to drought stress by adding a 10% polyethylene glycol (PEG) 6000 solution (Ahmad et al. 2021. Plant Physiol. Biochem. 166, 985–998) for durations of 6, 12 and 24 h. After 24 h, all leaves from both control and treated seedlings were collected simultaneously. Each sample consisted of three individual plants per biological replicate, with a total of three biological replicates per group. All samples were immediately frozen in liquid nitrogen and then stored at –80°C for subsequent analysis. Based on comments, the missing important details have been supplemented as follows.

(1) The Hoagland solution was used at 100× concentration in this study. In line 74: the concentration of Hoagland “ 100×” was added. Since the maximum treatment duration was 24 hours, no renewal or aeration was performed in this study. 

(2) In lines 77–80 “Leaf samples from both control and treatment groups were collected respectively for physiological index determination and transcriptome analysis with three biological replicates per group, The samples were immediately frozen in liquid nitrogen and stored at -80°C.” was revised as “After 24 h, all leaves from both control and treated seedlings were collected simultaneously. Each sample consisted of three individual plants per biological replicate, with a total of three biological replicates per group. All samples were immediately frozen in liquid nitrogen and then stored at –80°C for subsequent analysis.”

(3) As mentioned in the manuscript, all materials were cultivated uniformly until the seedlings reached the three-leaf-one-heart stage, after which the control and treatment groups were subjected to experimental conditions.

(4) We have referred to a large number of literature and set up polyethylene glycol (PEG) concentration gradients (5%, 10%, 15%, 20%) to conduct a pre-experiment on the Chinese cabbage seedlings at the three-leaf-one-heart stage, and finally determined the 10% PEG concentration.

According to comments, we have added a reference for Brassica species using this concentration on line 76 of the revised manuscript, and the corresponding citation has been included in the references section as follow.

In References section, “Ahmad, J.; Ali, A.A.; Al-Huqail, A.A.; Qureshi, M.I. Triacontanol attenuates drought-induced oxidative stress in Brassica juncea L. by regulating lignification genes, calcium metabolism and the antioxidant system. Plant Physiol. Biochem. 2021, 166, 985–998.” were added.

Comments 4: 2.2. Determination of Physiological Indicators

(1) Bibliographic references for the methods are missing, even though they are standard. For example: “according to [author, year] with minor modifications.”

(2) Measurement units are not provided (e.g., enzyme activity in U·mg⁻¹ protein, MDA in µmol·g⁻¹ FW).

(3) The measurement instrument and wavelength are not described.

(4) The number of technical replicates per sample is not mentioned.

(5) It is not indicated whether normalization was performed based on fresh weight or total protein.

Response 4: Thank you for your valuable comments on the "Determination of Physiological Indicators" section. We have made the following revisions as suggested.

In lines 82–86: “Superoxide dismutase (SOD) activity was determined using the nitroblue tetrazolium (NBT) photoreduction method; peroxidase (POD) activity was measured via the guaiacol method; malondialdehyde (MDA) content was analyzed using the thiobarbituric acid (TBA) colorimetric method; proline (Pro) content was quantified via the sulfosalicylic acid-acid ninhydrin colorimetric method.” was revised as “Superoxide dismutase (SOD, U/g FW) activity was determined using the photochemical reduction method of nitroblue tetrazolium (NBT) according to Dhindsa et al. [41] with absorbance measured at 450 nm. Peroxidase (POD, U/g FW) activity was measured using the guaiacol method as described by Hammerschmidt et al. [42] at 470 nm. Malondialdehyde (MDA, mmol/g) content was assessed via the thiobarbituric acid (TBA) colorimetric method following Guidicelli et al. [43] with measurements taken at 532 nm and 600 nm. Proline (Pro, μg/g) content was determined using the sulfosalicylic acid-acidic ninhydrin colorimetric method based on Bates et al. [44] at 470 nm. All detections were performed with three biological replicates.”

In References section,

“41. Dhindsa, R.S.; Matowe, W. Drought tolerance in two mosses: correlated with enzymatic defence against lipid peroxidation. J. Exp. Bot. 1981, 32, 79–91.

42. Hammerschmidt, R.; Nuckles, E.M.; Kuć, J. Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiol. Plant Pathol. 1982, 20, 73–82.

43. Guidi, L.; Bongi, G.; Ciompi, S.; Soldatini, G.F. In Vicia faba leaves photoinhibition from ozone fumigation in light precedes a decrease in quantum yield of functional PSII centres. J. Plant Physiol. 1999, 154, 167–172.

44. Bates, L.S.; Waldren, R.P.; Teare, I.D. Rapid determination of free proline for water-stress studies. Plant Soil. 1973, 39, 205–207.” were added.

Comments 5: 2.3. RNA Extraction, Illumina Sequencing, and Identification of DEGs

(1) The kit or protocol used for library construction, such as the TruSeq RNA Sample Prep Kit, is not specified.

(2) The sequencing platform, such as HiSeq, NovaSeq, or PE150, is not mentioned; it only states “Illumina sequencing.”

(3) There is no mention of RNA quality control (RIN values, concentration).

(4) The phrase “The total RNA was equivalently pooled with three replicates” is ambiguous and suggests that replicates were mixed, reducing statistical power. This should be clarified.

Response 5: Thank you for your constructive comments on the "RNA Extraction, DNB Sequencing, and Identification of DEGs" section. We have addressed each point as follows.

In lines 88–93: “Total RNA from T065-1 (CK, 6 h, 12 h and 24 h) and T065-2 (CK, 6 h, 12 h and 24 h) Chinese Cabbage leaves were individually extracted using Trizol reagents (Invitrogen, USA) following the manufacturer's instruction. The total RNA was equivalently pooled with three replicates and used for library preparation. A total of 24 cDNA libraries were constructed and sequenced. RNA-Seq data were deposited in NCBl Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/Traces/sra/).” was revised as

“Total RNA was individually extracted from T065-1 (CK, 6 h, 12 h, and 24 h) and T065-2 (CK, 6 h, 12 h, and 24 h) Chinese cabbage leaves using Trizol reagents (Invitrogen, USA) following the manufacturer's instructions. RNA quality was assessed using the Qubit 4.0 fluorometer and the Qsep400 bioanalyzer with RNA concentration ranged from 39 to 93 ng/μL. A total of 24 cDNA libraries were constructed following standard protocols from the eight samples with three biological replicates per sample. The libraries were initially quantified using Qubit 2.0 fluorometer, their insert sizes were determined using Agilent 2100 Bioanalyzer and quantified via Q-PCR method. The cDNA libraries were sequenced on the DNB sequencing platform by Metware Biotechnology Co., Ltd. (Wuhan, China). All raw RNA-Seq data were deposited in NCBI database (https://www.ncbi.nlm.nih.gov) with BioProject accession number PRJNA1337933.”

RNA Quality Inspection Results Table

Comments 6: 2.4. Identification of Key Candidate Genes

(1) This subsection is conceptual mainly and lacks detail on how “key candidate genes” were defined.

(2) The software or scripts used and the selection thresholds (e.g., top N genes by FDR or fold change) are not indicated.

(3) The method used for functional annotation validation (e.g., Blast2GO, EggNOG) is not mentioned.

Response 6: Details on key gene definition, used software, and annotation methods were provided as follows.

The screening criteria for DEGs were |log₂Fold Change| ≥ 1 and FDR < 0.05.

Genes were aligned to the NR, Swiss-Prot, TrEMBL, GO, KOG, and KEGG databases using DIAMOND software with an E-value threshold of < 1e-5. For Pfam annotation, protein sequences were aligned to the Pfam database via HMMER’s hmmscan tool, with an E-value threshold set at < 0.01. For transcription factor (TF) annotation in this plant study, the iTAK software was used, applying an E-value threshold of < 1e-5. Software versions: DIAMOND v2.0.9, hmmscan v3.2, iTAK 1.7a.

Comments 7: 2.5. qPCR Validation of DEGs

(1) The qPCR platform or equipment is not stated.

(2) Annealing temperature, number of cycles, and reaction volume are missing.

(3) There is no mention of amplification efficiency or product specificity (melting curve or single-band verification).

(4) A methodological reference for the 2–ΔΔCt method should be cited.

(5) The table does not show the amplicon sizes.

Response 7: Thank you for your valuable comments on the "qPCR Validation of DEGs" section. We have supplemented the relevant details as follows.

In response to comments (1), (2) and (4), we have supplemented the corresponding content in  lines 105–107: “Total RNA was extracted using the Trizol reagent (Invitrogen, USA) according to the manufacturer's protocol. Each sample was assayed in three technical and three biological replicates. qRT-PCR was conducted on a MyiQ Real-Time PCR detection system platform (Bio-Rad, USA) using the SYBR® Green Master ROX (TaKaRa, China). Each reaction was prepared in a total volume of 20 μl reaction mixture containing 2.0 ul of diluted cDNA, 0.2 μM primer pairs, and 10 ul of 2 × SYBR Green PCR Master Mix. The PCR reactions were carried out with the following program: 95 ℃ for 3 min, 40 cycles of 95 °C for 5 s, 58 °C for 30 s, and 72 ℃ for 10 s [46]. Specific primers were designed for 9 DEGs (Table 1). The relative expression level was quantified using the 2−ΔΔCT method with G6PD gene as the reference gene [47]. Statistical significance was determined by Duncan’s multiple range test at the P < 0.05 level using SPSS 21.0 statistical software (SPSS Inc., USA).”

In References section,

“46. Wang, R.; Mei, Y.; Xu, L.; Zhu, X.; Wang, Y.; Guo, J.; Liu, L. Differential proteomic analysis reveals sequential heat stress-responsive regulatory network in radish (Raphanus sativus L.) taproot. Planta. 2018, 247, 1109–1122.

47. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔC^T method. Methods. 2001, 25, 402–408.” were added.

In response to comments (3), to ensure amplification efficiency and product specificity, after extracting RNA and reverse transcribing it into cDNA, we conducted a roughly validation of the synthesized primer pairs as shown in Figure.S1. A total of nine primer pairs with proven efficacy and specificity were selected for further validation. These results will not be further elaborated in the manuscript.

Figure.S1 Agarose gel electrophoresis results of primer amplification

In response to comments (5), We have added a column labeled "fragment length (bp)" after the "Primer information" column in the original table 1 and filled in the corresponding amplicon length data for each primer pair.

Comments 8: Results

The authors obtained promising results; however, the text lacks explicit statistical analyses (e.g., ANOVA, p-values), and data repetition occurs. For example, “T065-1 had 3056 DEGs…” is mentioned in several forms.

Moreover, exact statistical values (p, logFC, FDR) for key genes are not reported. While the figure descriptions (Figures 2, 4, 6) are clear, they do not include quantitative data (e.g., magnitude of expression changes).

Response 8: Thank you for pointing out the issues related to statistical analysis and data presentation in the Results section. We have addressed these concerns as follows:

The statistical analysis descriptions in line 95: “The Benjamin-Hochberg method was used to correct the multiple hypothesis test probability (P value) to obtain the false discovery rate (FDR).” was supplemented. We have also removed redundant data descriptions to ensure manuscript conciseness.

For key genes (e.g., those involved in wax biosynthesis), we have added a supplementary table named “Table S1” to report their exact statistical values, including p-values, logFC, and FDR.

Comments 9: Discussion

The discussion is well structured and provides depth, but it tends to restate results rather than analyze them critically. This is particularly true in the first part, where it repeats which genes were expressed without explaining why.

Few direct functional comparisons are made with Brassica rapa or other crucifers; most parallels are drawn with Arabidopsis or rice.

Response 9: Thank you for your constructive comments on the Discussion section. Your suggestions have helped us improve the depth of analysis and relevance of comparative discussions, and we have revised the section as follows:

To address the issue of "restating results rather than critical analysis," we have revised the first part of the Discussion. We also link these gene expression patterns to physiological data to strengthen the logical connection between molecular findings and phenotypic traits.

In line 307 “ Non-waxy T065-2 showed earlier drought damage such as curled leaves and higher MDA, triggering more DEGs for stress response. By contrast, waxy T065-1 delayed stress damage, resulting in a milder gene response. These findings was consistent with the previously reported gene expression trends in Brassica napus under drought stress [51].”

In References section, “Lu, G.; Tian, Z.; Chen, P.; Liang, Z.; Zeng, X.; Zhao, Y.; Li, C.; Yan, T.; Hang, Q.; Jiang, L. Comprehensive morphological and molecular insights into drought tolerance variation at germination stage in Brassica napus accessions. Plants. 2024, 13, 3296.” was added.

Comments 10: Conclusions

This section is clear and correctly aligned with the objectives, listing key genes. However, it only summarizes results without discussing the broader implications of the research. It would strengthen the paper to include the practical implications of these findings in agriculture, beyond Chinese cabbage specifically. This could also be discussed in the Discussion section, highlighting the field-level impact and study limitations.

Response 10: Thank you for your suggestion on expanding the Conclusions section. We have revised it as follow:

In line 409: “This study provides critical technical support and application directions for addressing drought stress and ensuring stable development of the Brassicaceae vegetable industry.” was added.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response: Thank you for acknowledging that the English language quality of this manuscript meets the requirements. We will maintain rigorous language expression in subsequent revisions to uphold this quality standard.

5. Additional clarifications

Response: Additional clarifications on details were provided in the revised manuscript.

Response to Reviewer 3 Comments

1. Summary

Thank you for your insightful feedback. We appreciate your recognition of the study's relevance and structure, and we carefully addressed the methodological and data presentation suggestions to enhance the scientific rigor and reproducibility of our work.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

According to the corresponding comments, we have detailed revised this section in the point-by-point response letter as follows.

Is the research design appropriate?

Yes

Are the methods adequately described?

Can be improved

According to the corresponding comments, we have detailed revised this section in the point-by-point response letter as follows.

Are the results clearly presented?

Can be improved

According to the corresponding comments, we have detailed revised this section in the point-by-point response letter as follows.

Are the conclusions supported by the results?

Can be improved

According to the corresponding comments, we have detailed revised this section in the point-by-point response letter as follows.

Are all figures and tables clear and well-presented?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Abstract

The abstract is clear and concise.

I recommend briefly mentioning the experimental setup of the drought stress application, specifying whether it was withholding irrigation, PEG treatment, or soil-controlled deficit, to contextualize the transcriptomic findings.

Response 1: Thank you for your positive feedback on the abstract. We added a brief description of the drought stress experimental setup to the abstract as follow.

In lines 11–13: “this study conducted a comparative analysis of phenotypes, physiology and transcriptomes under drought stress using a pair of waxy near-isogenic lines (NILs) in Chinese cabbage.” was revised as “a pair of waxy near-isogenic lines (NILs) of Chinese cabbage were used as materials, and 10% polyethylene glycol (PEG) 6000 solution was employed to simulate drought stress. A comparative analysis of phenotypes, physiology and transcriptomes under drought stress was conducted in this study.”

Comments 2: Introduction

The introduction is informative but would benefit from expanded scientific context.

Please include references related to RNA-seq strategies for DEG detection under abiotic stress, particularly emphasizing de novo vs. reference-based transcriptome assembly approaches. Suggested references: https://doi.org/10.1016/j.stress.2024.100657; https://doi.org/10.3389/fgene.2022.958217;

Response 2: Thank you for your suggestion. We have expanded the introduction’s scientific context and added relevant RNA-seq references as follow.

In line 62: “Transcriptomic analysis is a powerful tool that enables researchers to identify genes and pathways activated or repressed during plant stress response [37–38]. With the long-read advantage of third-generation sequencing technology, assembly quality of the Chinese cabbage genome has been significantly improved, and the gene annotation of Chiifu V3.5 is more accurate and comprehensive.” was added.

In line 62: “This study used a pair of waxy NILs ......” was revised as “In this study, a reference-based transcriptomic approach was employed for a pair of waxy NILs......”

In References section,

“37. Privitera, G.F.; Treccarichi, S.; Nicotra, R.; Branca, F.; Pulvirenti, A.; Piero, A.R.L.; Sicilia, A. Comparative transcriptome analysis of B. oleracea L. var. italica and B. macrocarpa Guss. genotypes under drought stress: de novo vs reference genome assembly. Plant Stress. 2024, 14, 100657.

38. Singh, K.P.; Kumari, P.; Yadava, D.K. Development of de-novo transcriptome assembly and SSRs in allohexaploid Brassica with functional annotations and identification of heat-shock proteins for thermotolerance. Front Genet. 2022, 13, 958217.”were added.

Comments 3: Materials and Methods

The NCBI submission ID / BioProject or SRA accession number for the raw sequencing data must be included in this section and indicated again under Data Availability to ensure traceability and reproducibility.

Response 3: Thank you for your helpful comment on the "Materials and Methods" section. NCBI submission ID (SUB15676177) and BioProject accession number (PRJNA1337933) were acknowledged for the raw sequencing data in the "Materials and Methods" section as required.

In lines 92–93: “RNA-Seq data were deposited in NCBI Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/Traces/sra/).” was revised as “All raw RNA-Seq data were deposited in NCBI database (https://www.ncbi.nlm.nih.gov) with BioProject accession number PRJNA1337933.”

Comments 4: Results

Figures 7 and 8: Error bars should be added to the bar plots to indicate variability (standard error or standard deviation). The figure captions should be expanded to clearly explain the number of biological replicates, statistical test used, and meaning of any letters or symbols.

Lines 112–118: Please clarify whether phenotypic variations under drought were quantified using objective indicators, such as the SPAD index, the colorimetry or chlorophyll content; leaf lamina length, leaf area, relative water content (RWC), or digital morphometric analysis.

If these measurements were not taken, I strongly suggest including at least one morphometric comparison between drought-stressed and control plants to complement transcriptomic data with phenotypic evidence.

Response 4: Thank you for your valuable comments on the Results section. These suggestions have helped improve the completeness and scientific rigor of our data presentation.

For the phenotypic quantification indicators mentioned in lines 112–118, we have supplemented the description to clarify that phenotypic variations under drought stress were quantified using leaf relative water content (RWC). Detailed measurement methods and data for these indicators have also been added as follows.

In Materials and Methods section:

“2.2 Relative water content

Relative water content (RWC) was assessed using the first true leaf, with 12 biological replicates, for different materials under various drought stress conditions.The RWC was determined using the following formula: RWC (%) = [(FW-DW)/(TW-DW)] × 100, where FW = fresh weight, DW = dry weight, and TW = turgid weight [40].” was add before line 81.

In Results section:

“At 6 h of drought stress, RWC of the waxy T065-1 was lower than that of the non-waxy T065-2. As the drought stress duration extended to 12 h and 24 h, the RWC of waxy T065-1 became higher than that of non-waxy T065-2 (Figure 1c).” was add before line 119.

In References section: “Schonfeld, M.A.; Johnson, R.C.; Carver, B.F.; Mornhinweg, D.W. Water relations in winter wheat as drought resistance indicators. Crop Sci. 1998, 28, 526–531.” was add.

Additionally, other details have been revised in the revised manuscript.

Comments 5: Discussion

The discussion is well written and coherent.

Response 5: Thank you very much for your positive feedback on the "Discussion" section. During the revision process, we preserved the logical coherence of this section while refining certain expressions to further enhance the clarity of the arguments.

4. Response to Comments on the Quality of English Language

Point 1: The English is fine and does not require any improvement.

Response: Thank you for acknowledging that the English language quality of this manuscript meets the requirements. We will maintain rigorous language expression in subsequent revisions to uphold this quality standard.

5. Additional clarifications

Response: Additional clarifications on details were provided in the revised manuscript.