MdNAC17 Enhances Saline–Alkali Tolerance in Apple by Regulating Reactive Oxygen Species Removal

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

please see attached 

Comments for author File: Comments.pdf

Author Response

Dear Editors and Reviewers,

Thank you for your kind work and for the reviewers' comments concerning our manuscript entitled "MdNAC17 enhances saline-alkali tolerance in apple by regulating reactive oxygen species removal". Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction. Revised portion are marked in red in the paper. The main corrections in the paper and the responses to reviewers' comments are asas follows:

1. L25-29: Simplify the sentence and remove redundancy.

Response: Thank you very much for the reviewers' comments. I have condensed the sentences from L25-29 as follows: "Soil salinization can damage plant cell membranes, reduce enzyme functions, disrupt ion balance, leading to ion toxicity, osmotic stress and high pH damage."

2. L29-31: Shorten or better integrate the list of ions (Na⁺, Cl⁻, HCO₃⁻,CO₃²⁻).Shorten or better integrate the list of ions (Na⁺, Cl⁻, HCO₃⁻, CO₃²⁻).

Response: Thank you very much for the reviewers' comments. I have revised the sentence from L292-31 as follows: "For instance, under saline-alkali stress conditions, high concentrations of harmful ions (such as Na⁺, Cl^- and carbonate compounds) in the soil will inhibit the root system's absorption of essential nutrients like K⁺, Ca²⁺ and Mg²⁺."

3. L31-33:dd clearer mechanistic link or supporting references fophotosynthesis/PSII effects.

Response: Thank you very much for the reviewers' comments. I have revised the sentence from L31-33 to: "This will lead to a decrease in the conductance of leaf stomata, thereby reducing the supply of CO₂ to chloroplasts, resulting in a decline in photosynthetic activity and a decrease in the photochemical efficiency of PSII. Eventually, this will impede the growth and development of the crop."

4. L33-38: Condense general statements on adaptive strategies (ROS scavenging, gene expression).

Response: Thank you very much for the reviewers' comments. I have rephrased the sentences from L33 to L38 and revised them as follows:"To cope with the osmotic stress and ionic toxicity caused by saline-alkali soil, plants have evolved various adaptive strategies. For instance, plants activate the ROS clearance system and regulate the expression of stress-related genes to deal with osmotic stress and ionic toxicity, thereby reducing ion accumulation and ensuring their survival and reproduction."

5. L40-42: Improve transition toward NAC transcription factors.

Response: Thank you very much for the reviewers' comments. I have revised the sentences from L40 to L42 and changed them to "The regulation of stress response genes in plants is primarily mediated by transcription factors (TFs). By modulating gene expression, plants can adapt to or mitigate the impacts of stress conditions, thereby supporting crop development and enhancing yield^[11]. NAC transcription factors (TFs) are a major plant-specific family defined by a conserved N-terminal DNA-binding domain, a nuclear localization signal, and a variable C-terminal transcriptional activation domain."

6. L42-47: Reduce or better focus examples not directly related to saline-alkali stress.

Response: Thank you very much for the reviewers' comments. We have removed the examples that were not directly related to salt and alkali stress, and have added new examples instead: "For example, in tomato (Solanum lycopersicum L.), SlNAC2 promotes melatonin accumulation and ROS clearance by partially counteracting the inhibitory effects on SlCOMT2 and SlSNAT. SlNAC63, on the other hand, enhances the regulation of SlAOS1 and SlSOD4 to promote jasmonic acid accumulation and ROS clearance. These two NAC genes jointly enhance the salt-tolerance and alkaline resistance of tomato."

7. L48-51:Clarify relevance of ABA/NAC examples to salt stress response.

Response: Thank you very much for the reviewers' comments. I have revised the sentence from L48-51 and changed it to: "Under adverse stress conditions, the ABA signaling pathway is activated, which then regulates the expression of some NAC transcription factors to enhance the plant's stress resistance. For instance, the expressions of ANAC072, ANAC055 and ANAC019 in Arabidopsis are induced by high salinity, drought, ABA and MeJA; while AtNAC016 enhances the drought resistance by inhibiting the negative regulatory factor AREB1 of the ABA signaling pathway. These mechanisms collectively reveal the important regulatory role of NAC transcription factors in the response to salt stress."

8. L51-54:trengthen link between NAC functions and saline-alkali stress.

Response: Thank you very much for the reviewers' comments. I have revised the sentences from L51 to L54 and changed them to "Although it is known that NAC transcription factors play a significant role in plant growth, development, and responses to non-biological stresses such as salinity and alkalinity, the specific molecular regulatory mechanism of these factors under salt and alkaline stress, especially in apples, still needs to be further elucidated."

9. L63-66: Make MdNAC17introduction more concise.

Response: Thank you very much for the reviewers' comments. I have revised the sentences from L63 to L66 and changed them to "However, the functions of most of the NAC genes in apples remain unknown. The mechanism of action of MdNAC17 under saline-alkali stress has not yet been clarified. Only preliminary mentions have been found, and there is a lack of systematic functional verification."

10. L66-68: clarify the objective statement.

Response: Thank you very much for the reviewers' comments. I have revised the sentences from L66 to L68 and changed them to " Therefore, this study aimed to explore the function of MdNAC17 in apple saline-alkali tolerance, thereby providing a theoretical basis for genetically improving saline-alkali resistance."

11. L72: Clarify further the choice of Malus hupehensis and its relevance to saline-alkali stress tolerance studies

Response: Thank you very much for the reviewers' comments. I have revised the sentences from L66 to L68 and changed them to "Selecting Malus hupehensis (Pamp.) Rehd. var. pingyiensis Jiang, which has strong salt-alkali resistance, for gene expression analysis and genetic transformation."

12. L76-79: rovide additional details on seedling growth conditions (light intensity, photoperiod, humidity).

Response: Thank you very much for the reviewers' comments. The relevant detailed information has been added as Place it in a culture environment at 25℃, with a light-dark ratio of 16/8 and 80% humidity.".

13. L81: Add more details on in vitro culture conditions (light conditions, dark incubation if applied).

Response: Thank you very much for the reviewers' comments. Relevant detailed information has been added as "Thank you again for the reviewers' comments. It has been stated in the text that the apple callus was cultivated in the dark under an ordinary light incubator, with a cultivation temperature of 25℃. "

14. L83:ndicate whether the subculturing interval (20 days) is standard or optimized for this system

Response: We would like to express our sincere gratitude to the reviewers for their valuable comments. It has been clearly stated in the text that the system's standard setting is to subculture the callus every 20 days.

15. L250:Improve the quality of all figures/images to ensure higher resolution, better readability, and clearer visualization of data

Response: Thank you very much for the reviewers' comments. I have re-adjusted all the pictures in the article, aiming to enhance their resolution and readability as much as possible.

16. L417–420: The opening sentence is a bit too general and could be slightly shortened to be more direct.

Response: Thank you very much for the reviewers' comments. I have reorganized L417-420 and changed it to: "Saline-alkali stress is a significant abiotic stress that restricts apple production. NAC transcription factors, as key regulatory nodes in stress responses, the mechanism by which they mediate apple's tolerance to saline-alkali conditions deserves in-depth exploration."

17. L420–423: The transition into NAC transcription factor examples could be smoother and less repetitive.

Response: Thank you very much for the reviewers' comments. I have inserted "In fact, the NAC transcription factors have diverse functions in regulating plant stress tolerance. They even exhibit opposite effects in different species and under different stress conditions." before paragraphs L420-423.

18. L423–426: The OsNAC10and OsNAC2examples are relevant, but the explanation could be simplified and more directly linked to oxidative and ion stress.

Response: Thank you very much for the reviewers' comments. I have revised the paragraphs 423-426 as follows: "For instance, in rice (Oryza sativa L.), overexpression of OsNAC10 enhanced drought resistance;" while OsNAC2 inhibited stress-related genes (such as OsLEA3, OsSAPK1), intensified oxidative and ionic damage, thereby reducing drought resistance and salt tolerance."

19. L426–430: The transition from literature background to the authors’ results could be made clearer.

Response: Thank you very much for the reviewers' comments. I have revised the paragraphs 426-430 to "In soybeans (Glycine max), the overexpression of GmNAC06 can enhance leaf salt tolerance by eliminating ROS and regulating Na⁺/K⁺ transport, thereby maintaining ion and osmotic balance ^[45]. Similarly, in this study, it was found that apple MdNAC17 was significantly upregulated under saline-alkali treatment (Figure 2). Its overexpression can enhance the salt and alkali tolerance of callus tissue and apple transgenic roots, while the TRV-MdNAC17 silenced plants showed greater sensitivity. This indicates that MdNAC17 is a positive regulatory factor for apple's salt and alkali tolerance."

20. L431–436: The section on chlorophyll content is somewhat repetitive and could be condensed.

Response: Thank you very much for the reviewers' comments. I have condensed the paragraphs L431-436 into "Chlorophyll is the main photosynthetic pigment, but it is prone to decomposition under stress conditions, thereby reducing photosynthetic efficiency. This study found that the chlorophyll content in apple leaves under saline-alkali stress was generally decreased, but the chlorophyll content in MdNAC17-OE plants was significantly higher than that in TRV-MdNAC17 and EV plants (Figures 5b, 8b), indicating that MdNAC17 maintains a higher photosynthetic efficiency by inhibiting chlorophyll degradation."

21. L436–441: The description of photosynthetic parameters (Fm, qP, NPQ, etc.) is quite dense and could be simplified for better readability.

Response: We are very grateful for the reviewers' comments. We have reorganized the paragraphs 436-441 and revised them as "The subsequent results further support this hypothesis: the parameters related to photochemical efficiency (Fv/Fm, Y(II), Fm, qP) in the leaves of MdNAC17-OE plants, as well as the parameters related to energy dissipation (qN, YNPQ, NPQ/4), were significantly higher than those of the EV plants, while the TRV-MdNAC17 plants were significantly lower than the EV plants (Figures 5 and 8). It is well known that qP reflects the proportion of light energy used for electron transfer in PSII, while NPQ and qN are used to dissipate excess light energy, thereby actively avoiding photoinhibition and oxidative damage in PSII. These results indicate that the overexpression of MdNAC17 enhances the tolerance to salt and alkali by increasing the efficiency of photochemical electron transfer and reducing the thermal dissipation demand of excess energy."

22. L441–444: The physiological interpretation of NPQ and qN could be expressed more clearly and directly.

Response: Thank you very much for the reviewers' comments. I have now re-explained L441-444 in a clearer and more direct manner, changing it to "It is well known that qP reflects the proportion of light energy used for electron transfer in PSII, while NPQ and qN are used to dissipate excess light energy, thereby actively avoiding photoinhibition and oxidative damage in PSII. These results indicate that the overexpression of MdNAC17 enhances the tolerance to salt and alkali by increasing the efficiency of photochemical electron transfer and reducing the thermal dissipation demand of excess energy."

23. L446–450: The sentences describing ROS, MDA, and REC show some repetition andcould be merged.

Response: Thank you very much for the reviewers' comments. I have revised this paragraph and changed it to "Under salt and alkali stress, excessive accumulation of ROS can lead to membrane lipid peroxidation (MDA) and increased membrane permeability (REC). In this study, MdNAC17-OE significantly reduced the levels of H₂O₂ and O₂^-, while TRV-MdNAC17 resulted in significantly increased MDA and REC (Figures 4 and 7), indicating that MdNAC17 alleviates oxidative membrane damage by reducing ROS accumulation."

24. L450–452: The conclusion about osmotic balance is valid but could be stated more concisely.

Response: Thank you very much for the reviewers' comments. I have made the necessary revisions to it as "These results indicate that MdNAC17 plays a positive role in maintaining osmotic balance and mitigating membrane damage".

25. L452–455: The antioxidant enzyme section is solid but slightly long and could be shortened.

Response: Thank you very much for the reviewers' comments. I have condensed this section into "To cope with oxidative damage, plants have developed a series of complex protective mechanisms, such as activating antioxidant enzymes to eliminate ROS. In this study, under saline-alkali stress conditions, the expression levels of MdSOD, MdPOD, and MdCAT in apple MdNAC17-OE plants and their corresponding enzyme activities were significantly higher than those in the control group (Figure 6; Figure 4j-l), suggesting that MdNAC17 enhances the antioxidant defense system, promotes the metabolism of reactive oxygen species, and thereby improves the salt-alkali tolerance of plants, which is consistent with the research results of Wang L. et al. (2017)."

26. L455–459: There is some redundancy between gene expression and enzyme activity results, which could be streamlined.

Response: Thank you very much for the reviewers' comments. I have condensed this section into "To cope with oxidative damage, plants have developed a series of complex protective mechanisms, such as activating antioxidant enzymes to eliminate ROS. In this study, under saline-alkali stress conditions, the expression levels of MdSOD, MdPOD, and MdCAT in apple MdNAC17-OE plants and their corresponding enzyme activities were significantly higher than those in the control group (Figure 6; Figure 4j-l), suggesting that MdNAC17 enhances the antioxidant defense system, promotes the metabolism of reactive oxygen species, and thereby improves the salt-alkali tolerance of plants, which is consistent with the research results of Wang L. et al. (2017)."

27. L461–465: The role of proline is well explained but slightly overstated and should be more balanced.

Response: Thank you very much for the reviewers' comments. I have condensed this section into "This result suggests that overexpression of MdNAC17 in the roots may be related to the maintenance of osmotic balance in the above-ground non-transgenic leaves and the alleviation of membrane damage. One possible explanation is that MdNAC17 positively regulates the biosynthesis of osmotic regulatory substances such as proline in the roots, thereby contributing to the overall tolerance of plants to saline-alkali stress. However, the specific systematic signaling pathways still require further study."

28. L467–470: The description of the SOS pathway is clear but could be simplified for smoother reading.

Response: Thank you very much for the reviewers' comments. I have condensed this section into "The ionic toxicity caused by salt and alkali stress can inhibit the growth and development of plants. In this study, when MdNAC17 was overexpressed or subjected to stress treatment, the transcriptional levels of key genes in the SOS signaling pathway, as well as genes related to Na⁺ excretion (MdNHX1, MdHKT1) and proton pump genes (MdAHA2, MdAHA8), all showed an upward trend."

29. L470–473: The Na⁺/K⁺ homeostasis section is important but somewhat dense and could be made more concise.

Response: Thank you very much for the reviewers' comments. I have condensed this section into " Furthermore, the transcriptional level of the K⁺ efflux channel gene MdGORK1 has decreased."

30. L473–475: The link between transport genes and physiological outcomes should be more clearly emphasized.

Response: Thank you very much for the reviewers' comments. I have revised this paragraph to " These changes indicate that MdNAC17 may enhance Na⁺ excretion and compartmentalization ability by upregulating the expression of SOS pathway proteins and H⁺-ATPase-related genes, thereby maintaining the ionic homeostasis within the cells."

31. L476–479: The ABA-related interpretation is interesting but should be presented with more caution unless experimentally confirmed.

Response: Thank you very much for the reviewers' comments. I have revised this paragraph and changed it to " On the other hand, NAC transcription factors have been widely reported to be involved in non-biological stress responses through the ABA pathway. This study found that the promoter region of MdNAC17 contains ABA response elements, and in the transgenic materials overexpressing MdNAC17, the expression of the key ABA pathway gene MdAREB1 was significantly upregulated."

32. L479–481: The final sentence is good but could be simplified for better clarity and impact.

Response: Thank you very much for the reviewers' comments. I have revised this paragraph and changed it to "These correlations suggest that MdNAC17 may be associated with the ABA signaling pathway and may be involved in coordinating the clearance of reactive oxygen species and the regulation of ionic homeostasis, but whether MdNAC17 directly regulates MdAREB1 or other ABA pathway genes still requires further experimental verification."

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors show that MdNAC17 expression is induced under saline-alkali stress and that overexpression improves callus and root performance under stress, while silencing reduces tolerance. The physiological results are consistent; overexpression is associated with higher antioxidant enzyme activities, lower MDA/REC, reduced ROS accumulation, and improved photosynthetic parameters under saline-alkali stress. The topic is relevant for apple rootstock improvement under saline/alkaline soil conditions.  However, several important issues should be addressed:

1/ Most mechanistic evidence is based on physiological measurements and RT-qPCR of stress-related genes. The observed increase in MdSOD, MdPOD, MdCAT, MdSOS1, MdNHX1, MdHKT1, MdAHA2, MdAHA8, MdSOS2, MdSOS3, and MdAREB1 supports an association, but it does not prove direct transcriptional regulation by MdNAC17.

2/ The authors should either add direct evidence, such as yeast one-hybrid, EMSA, ChIP-qPCR, or dual-luciferase assays, or revise the wording to “is associated with” or “may promote” ROS scavenging and stress-related gene expression.

3/ The manuscript predicts that MdNAC17 is localized in the endoplasmic reticulum, while NAC transcription factors generally function in the nucleus. This is a major conceptual problem because the manuscript argues that MdNAC17 regulates the expression of stress-responsive genes.

4/ The authors should experimentally validate the subcellular localization using MdNAC17-GFP transient expression, preferably with nuclear and ER markers. If MdNAC17 is truly ER-associated, the authors must explain how it functions as a transcription factor, for example through stress-induced cleavage or relocalization.

5/ The manuscript concludes that MdNAC17 maintains Na⁺/K⁺ homeostasis, but the data shown are mainly transcript levels of transporter-related genes. This is not sufficient to claim improved ion homeostasis. The authors should measure Na⁺ and K⁺ contents in roots and leaves, Na⁺/K⁺ ratios, and ideally H⁺-ATPase activity or Na⁺ efflux. Otherwise, statements such as “maintaining cellular Na⁺/K⁺ homeostasis” should be softened.

6/ The overexpression experiment in Malus hupehensis appears to involve transformed roots generated through Agrobacterium rhizogenes. However, several measured parameters are from leaves, including RWC, ROS staining, and chlorophyll fluorescence. The authors should explain whether the shoots were also transgenic or whether the observed leaf effects are indirect consequences of transgenic roots. This is important for interpreting whole-plant tolerance.

7/ MdNAC17 silencing reduced transcript abundance by about 69%, but the authors should show whether the 238 bp VIGS fragment is specific to MdNAC17. Potential off-target effects on closely related NAC genes should be checked by sequence comparison and, if possible, qPCR of homologous NAC members.

8/ The discussion mainly repeats the results and links them to general ROS, photosynthesis, and ion-homeostasis concepts. It should be improved by explaining why MdNAC17 may coordinate antioxidant defense, osmotic adjustment, photosynthetic protection, and ion transport. The authors should also discuss the study's limitations, particularly the lack of direct target validation and field- and rootstock-level validation.

9/ The discussion could be broadened by integrating evidence from other woody species adapted to arid and semi-arid environments (doi: 10.1016/j.jaridenv.2023.105095 ; 10.1016/j.bcab.2022.102457)

10/ Several figure panels are very small, especially phylogenetic trees, motif structures, fluorescence images, and multigraph panels. Scale bars, statistical letters, treatment abbreviations, and sample sizes should be clearly defined in each legend. Figure 9 is useful but too simplified; it should distinguish demonstrated results from proposed mechanisms.

Comments on the Quality of English Language

Should be improved

Author Response

Dear Editors and Reviewers,

Thank you for your kind work and for the reviewers' comments concerning our manuscript entitled "MdNAC17 enhances saline-alkali tolerance in apple by regulating reactive oxygen species removal". Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction. Revised portion are marked in red in the paper. The main corrections in the paper and the responses to reviewers' comments are asas follows:

1. 1.Most mechanistic evidence is based on physiological measurements and RT-qPCR of stress-related genes. The observed increase in MdSOD, MdPOD, MdCAT, MdSOS1, MdNHX1, MdHKT1, MdAHA2, MdAHA8, MdSOS2, MdSOS3, and MdAREB1supports an association, but it does not prove direct transcriptional regulation by MdNAC17.

Response: Thank you for this important comment. We fully agree that the observed upregulation of stress-related genes does not prove that MdNAC17 has a direct transcriptional regulatory effect. We have carefully revised the text to clearly state that the observed increase in the expression levels of these genes is correlated with MdNAC17, but it is not necessarily caused by it. However, at present, we lack direct evidence for most of these genes. We clearly state that the direct transcriptional regulatory mechanism still needs to be further verified. We have added the following sentence in the discussion section: "Although the overexpression of MdNAC17 leads to an increase in the transcriptional levels of multiple genes related to stress, whether MdNAC17 directly regulates these genes still needs to be further studied."

2. 2.The authors should either add direct evidence, such as yeast one-hybrid, EMSA, ChIP-qPCR, or dual-luciferase assays, or revise the wording to “is associated with” or “may promote” ROS scavenging and stress-related gene expression.

Response: Thank you for this important comment. We fully agree that the observed upregulation of stress-related genes does not prove that MdNAC17 has a direct transcriptional regulatory effect. We have carefully revised the text to clearly state that the observed increase in the expression levels of these genes is correlated with MdNAC17, but it is not necessarily caused by it. However, at present, we lack direct evidence for most of these genes. We clearly state that the direct transcriptional regulatory mechanism still needs to be further verified. We have added the following sentence in the discussion section: "Although the overexpression of MdNAC17 leads to an increase in the transcriptional levels of multiple genes related to stress, whether MdNAC17 directly regulates these genes still needs to be further studied."

3. 3.The manuscript predicts that MdNAC17is localized in the endoplasmic reticulum, while NAC transcription factors generally function in the nucleus. This is a major conceptual problem because the manuscript argues that MdNAC17 regulates the expression of stress-responsive genes.

Response: Thank you for raising this crucial conceptual question. We fully agree that the NAC transcription factor must be localized in the cell nucleus in order to regulate gene expression, and we also acknowledge that our initial interpretation of the WoLFPSORT prediction results (ER 3.5 vs. nucleus 3.0) was incorrect. To clearly address this issue, we expressed the MdNAC17-GFP fusion protein in tobacco leaves and conducted subcellular localization analysis. As shown in the newly added Figure 1h, the fluorescence of MdNAC17-GFP mainly detected the nucleus and co-localized with the nucleus Marker. This experimental result clearly indicates that MdNAC17 is a protein localized in the nucleus, which is consistent with its function as a transcription factor. We have revised the manuscript: removed the misleading ER localization statement based on WoLFPSORT; added the subcellular localization experiment (Figure 1h); and noted in the results section: "Subcellular localization analysis showed that MdNAC17 is localized in the nucleus (Figure 1h)." We thank the reviewers for this important verification opinion, which greatly enhanced the quality of our paper.

4. 4.The authors should experimentally validate the subcellular localization using MdNAC17-GFP transient expression, preferably with nuclear and ER markers. If MdNAC17is truly ER-associated, the authors must explain how it functions as a transcription factor, for example through stress-induced cleavage or relocalization.

Response: Thank you for raising this crucial conceptual question. We fully agree that the NAC transcription factor must be localized in the cell nucleus in order to regulate gene expression, and we also acknowledge that our initial interpretation of the WoLFPSORT prediction results (ER 3.5 vs. nucleus 3.0) was incorrect. To clearly address this issue, we expressed the MdNAC17-GFP fusion protein in tobacco leaves and conducted subcellular localization analysis. As shown in the newly added Figure 1h, the fluorescence of MdNAC17-GFP mainly detected the nucleus and co-localized with the nucleus Marker. This experimental result clearly indicates that MdNAC17 is a protein localized in the nucleus, which is consistent with its function as a transcription factor. We have revised the manuscript: removed the misleading ER localization statement based on WoLFPSORT; added the subcellular localization experiment (main text Figure 1h); and noted in the results section: "Subcellular localization analysis showed that MdNAC17 is localized in the nucleus (Figure 1h)." We thank the reviewers for this important verification opinion, which greatly enhanced the quality of our paper.

5. 5.The manuscript concludes that MdNAC17maintains Na⁺/K⁺ homeostasis, but the data shown are mainly transcript levels of transporter-related genes. This is not sufficient to claim improved ion homeostasis. The authors should measure Na⁺ and K⁺ contents in roots and leaves, Na⁺/K⁺ ratios, and ideally H⁺-ATPase activity or Na⁺ efflux. Otherwise, statements such as “maintaining cellular Na⁺/K⁺ homeostasis” should be softened.

Response: Thank you for raising this important and constructive suggestion. We fully agree with your viewpoint. Our current data mainly come from the transcriptional levels of genes related to ion transport proteins and cannot directly prove that the ion homeostasis or the maintenance of the Na⁺/K⁺ ratio have been improved. In the revised manuscript, we have carefully removed all such functional statements and replaced them with descriptive or correlational expressions consistent with the gene expression data. For example, "MdNAC17 maintains the Na⁺/K⁺ balance" has been changed to "Furthermore, MdNAC17 is associated with changes in the transcriptional levels of genes involved in Na+/K+ homeostasis." "MdNAC17 activates the SOS pathway..." has been changed to "These changes suggest that MdNAC17 may be associated with SOS pathway proteins and H⁺-ATPase in terms of regulation, and jointly participate in the maintenance of ionic homeostasis." We have also clearly pointed out in the revised discussion section that this is a limitation of this study: although our data indicate an association between the expression of MdNAC17 and the transcription of genes related to ion transport, the above correlation results are not sufficient to prove that MdNAC17 directly regulates these genes. To determine whether MdNAC17 truly improves ion homeostasis, direct measurements of Na⁺ and K⁺ concentrations, Na⁺/K⁺ ratios, and H⁺-ATPase activity, etc., are still needed. These measurement results provide important directions for future research. We appreciate the reviewers for insisting on using accurate and evidence-based language. We believe that the revised manuscript now accurately reflects the scope of our conclusions.

6. 6.The overexpression experiment inMalus hupehensis appears to involve transformed roots generated through Agrobacterium rhizogenes. However, several measured parameters are from leaves, including RWC, ROS staining, and chlorophyll fluorescence. The authors should explain whether the shoots were also transgenic or whether the observed leaf effects are indirect consequences of transgenic roots. This is important for interpreting whole-plant tolerance.

Response: Thank you for raising this important and constructive suggestion. We fully agree with your opinion. In our transformation experiment system mediated by the Agrobacterium root-knot gene, only the roots are transgenic, while the above-ground parts (including the leaves used for RWC, ROS staining and chlorophyll fluorescence measurement) are non-transgenic. Therefore, the physiological changes observed in the leaves, such as the changes in RWC, ROS levels and chlorophyll fluorescence, are the indirect effects caused by the transgenic roots. We have accordingly revised the paper to avoid any assertion about direct transgenic effects in the leaves.

7. 7.MdNAC17silencing reduced transcript abundance by about 69%, but the authors should show whether the 238 bp VIGS fragment is specific to MdNAC17. Potential off-target effects on closely related NAC genes should be checked by sequence comparison and, if possible, qPCR of homologous NAC members.

Response: Thank you for raising this key technical issue. We fully agree that verifying the specificity of the 238 bp VIGS fragment is of utmost importance. After comparing the MdNAC17 sequence in the apple genome GDR database, we found a homologous gene MD05G1037700, whose full-length identity in the coding region with MdNAC17 is as high as 93.75%. It is impossible to specifically interfere with either one of them, so we chose to interfere with both genes simultaneously.

8. 8.The discussion mainly repeats the results and links them to general ROS, photosynthesis, and ion-homeostasis concepts. It should be improved by explaining why MdNAC17 may coordinate antioxidant defense, osmotic adjustment, photosynthetic protection, and ion transport. The authors should also discuss the study's limitations, particularly the lack of direct target validation and field- and rootstock-level validation.

Response: Thank you for your valuable suggestions. We have revised the discussion section based on your opinions. Specifically, we have further clarified that MdNAC17 may be involved in antioxidant defense, osmotic regulation, and photoprotection mechanisms of photosynthesis. We have also added a discussion on the limitations of this study, such as the lack of direct verification and confirmation of the target gene under field conditions. The revised content has been highlighted in the updated manuscript. We sincerely thank the reviewers for their constructive comments.

9. 9.The discussion could be broadened by integrating evidence from other woody species adapted to arid and semi-arid environments (doi: 10.1016/j.jaridenv.2023.105095 ; 10.1016/j.bcab.2022.102457)

Response: We are very grateful for the reviewers' comments. We have added relevant content in the discussion section to further expand this discussion. The content is as follows: "Ait Hammou et al. (2023) reported that under a 102.6 mmol/L NaCl treatment, proline and carbohydrate metabolites in the seeds of Argania spinosa increased significantly, thereby enhancing the salt tolerance of the seeds. It is worth noting that this species naturally occurs in semi-arid environments, and its osmotic regulation mechanism may have evolved under the dual selection pressure of long-term drought and salt-alkali. In conclusion, our results further support that the osmotic regulation ability is one of the key mechanisms for woody plants to tolerate salt and alkali stress."

10. 10.Several figure panels are very small, especially phylogenetic trees, motif structures, fluorescence images, and multigraph panels. Scale bars, statistical letters, treatment abbreviations, and sample sizes should be clearly defined in each legend. Figure 9 is useful but too simplified; it should distinguish demonstrated results from proposed mechanisms.

Response: We sincerely thank you for your detailed and constructive comments on our chart. We have carefully revised each of your feedbacks and reorganized Figure 1, enlarged each individual panel as much as possible, deleted Figure 9 in accordance with the opinions of other reviewers, re-adjusted Figures 2-8 to ensure the annotation of the scale in the image, revised all the legends, and supplemented the missing information.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript submitted by Liu and colleagues investigated the role of a specific NAC-transcription factor from apple trees (NAC17) and its role in saline-alkaline stress. The authors overexpressed and silenced the gene and exposed the calli/seedlings to saline alkaline. They measured a number of parameters and expression of genes known to be related to abiotic stress. In brief, it was shown that plants overexpressing / silenced in the expression of NAC17 showed opposite effects, with higher stress tolerance in overexpressing lines.

The study is interesting and the experimental approach solid. I only have a few comments where the manuscript could be improved.

line 27 enzyme activities (not enzymes)

line 45 results in early ...

line 48 stress conditions

line 60 shown to be involved

line 66 Therefore, this study seeks

line 195 Virus-induced gene silencing in apple trees

line 351-353 This sentence and a brief description of the genes should be moved to the introduction.

Figure 9 can be deleted. It does not show a mechanism, but only describes what happens after overexpression and down-regulation of NAC17.

line 434 Delete This indicated that and start with: Saline-alkali stress

line 441 ...., while plants rapidly engage NPQ and qN....

Author Response

Dear Editors and Reviewers,

Thank you for your kind work and for the reviewers' comments concerning our manuscript entitled "MdNAC17 enhances saline-alkali tolerance in apple by regulating reactive oxygen species removal". Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction. Revised portion are marked in red in the paper. The main corrections in the paper and the responses to reviewers' comments are asas follows:

1. line 27 enzyme activities (not enzymes)

Response: Thank you very much for the reviewers' comments. I have made the necessary revisions based on their suggestions.

2. line 45 results in early ...

Response: We are very grateful for the reviewers' comments. After considering the opinions of other reviewers as well, this sentence has been deleted.

3. line 48 stress conditions

Response: Thank you very much for the reviewers' comments. I have revised it to "stress conditions".

4. line 60 shown to be involved

Response: Thank you very much for the reviewers' comments. Considering the opinions of other reviewers as well, it has been revised to "Among the NAC transcription factors identified in apples, certain members have been shown to be involved in abiotic stress responses."

5. line 66 Therefore, this study seeks

Response: Thank you very much for the reviewers' comments. Considering the opinions of other reviewers as well, I have revised it to: "Therefore, this study aimed to explore the function of MdNAC17 in apple's saline-alkali tolerance, thereby providing a theoretical basis for genetically improving saline-alkali resistance."

6. line 195 Virus-induced gene silencing in apple trees

Response: Thank you very much for the reviewers' comments. I have revised it to "virus-induced gene silencing (VIGS) in apple plants".

7. line 351-353This sentence and a brief description of the genes should be moved to the introduction.

Response: Thank you for the reviewers' suggestions. Considering the opinions of other reviewers, we have revised this sentence. Specifically, since the RT-qPCR analysis can only provide evidence of the correlation between MdNAC17 expression and the selected stress-related genes, but not a direct regulatory relationship, we believe it is inappropriate to elaborate on these genes in the introduction. Therefore, we retained this section in the results and made some minor adjustments, changing it to: "In order to further investigate the role of MdNAC17 in the regulation of apple root responses to saline-alkali stress, we used RT-qPCR to detect the expression levels of several stress-related genes. According to previous reports, the following genes were selected as candidate genes for saline-alkali stress responses."

8. Figure 9 can be deleted. It does not show a mechanism, but only describes what happens after overexpression and down-regulation of NAC17.

Response: Thank you very much for the reviewers' comments. Figure 9 has been removed.

9. line 434 Delete This indicated that and start with: Saline-alkali stress

Response: Thank you very much for the reviewers' comments. Considering the opinions of other reviewers as well, I have revised it to: "This study found that the chlorophyll content in apple leaves under saline-alkali stress was generally decreased."

10. line 441 ...., while plants rapidly engage NPQ and qN....

Response: Thank you very much for the reviewers' comments. I have made the necessary revisions to it as "It is well known that qP reflects the proportion of light energy used for electron transfer in PSII, while NPQ and qN are used to dissipate excess light energy, thereby actively avoiding photoinhibition and oxidative damage in PSII."

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept in present form

Reviewer 2 Report

Comments and Suggestions for Authors

All issues raised during the review process have been adequately addressed. The manuscript has been improved accordingly and may now be considered for publication in its current form.