Comparative Phenotype and Transcriptome Profiling in Some Grapevine Cultivars in Response to Drought Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents an investigation into the drought tolerance of 30 grapevine genotypes, combining phenotypic and transcriptomic analyses. While the study offers some interesting data, several significant drawbacks and minor issues need to be addressed before it can be considered for publication.

My Major concerns are:

- The introduction broadly discusses drought stress and grapevine responses but lacks a clear, focused objective statement or specific hypotheses that the study aims to test. This makes it difficult to assess the study's success in achieving its goals.

- The paper mentions that nine phenotypic traits were examined but does not adequately justify why these specific traits were chosen over others, or how their relevance to drought tolerance was established prior to the study. A more robust explanation of trait selection is needed.

- The transcriptomic analysis identified only 55 differentially expressed genes (DEGs) when comparing control and stressed groups across five cultivars. This number is remarkably low for a study of this nature and suggests either a very conservative analysis threshold, insufficient sequencing depth, or that the chosen stress duration/intensity was not optimal for capturing a broader transcriptomic response. This significantly limits the depth of molecular insights.

- Phenotypic analysis used 1–6% mannitol for one month, while transcriptomic analyses used 2% mannitol for 6–12 hours. These differences in concentration and duration complicate direct comparison and interpretation.

- The study identifies DEGs but does not include any experimental validation of these findings (e.g., using qPCR). Without validation, the reliability of the transcriptomic data remains questionable, especially given the low number of identified DEGs.

-  While the paper mentions general patterns under drought stress, such as antioxidant activation and changes in metabolism, the discussion does not sufficiently highlight novel findings or their broader implications for grapevine breeding or crop management.

- The paper states that ANOVA was performed for all parameters to estimate the significance of stress, cultivar, and their interaction, and means were separated using LSD at p ≤ 0.05. However, specific details about the ANOVA model (e.g., fixed versus random effects, interaction terms) and post-hoc tests are not fully elaborated for all analyses, which could impact the robustness of the phenotypic conclusions.

- While mannitol is a common osmoticum, its effects can differ from actual water deficit. The study does not sufficiently discuss the potential limitations of using mannitol to simulate drought stress and how this might influence the generalizability of the findings to field conditions.

Minor Issues:

-Typographical Errors and Grammatical Inconsistencies: There are several minor grammatical errors and awkward phrasings throughout the text (e.g., "grapevines generally shows an avoidance strategy" , "After one month of exposure, thirty grapevine genotypes showed different responses to drought". These should be corrected for clarity and professionalism.

- Some figures are referenced generally (e.g., "Fig. 1"), while others are very specific (e.g., "Fig. 1b, d and e").

- Some figures are difficult to read due to small font sizes and crowded information. Ensuring all figures are clear and legible is crucial.

Author Response

We thank the Reviewer very much for the careful reading of our manuscript and for providing useful comments.

We revised the manuscript in accordance with the comments of the Reviewer. Our comments and answers are given below.

REV: The introduction broadly discusses drought stress and grapevine responses but lacks a clear, focused objective statement or specific hypotheses that the study aims to test. This makes it difficult to assess the study's success in achieving its goals.

Author: Thank the Reviewer for this comment. We rewritten Introduction according to the Reviewer suggestion and added the study aims: «Studies have also been conducted on grapevines to investigate the effects of drought on plant physiology and to identify the molecular mechanisms underlying drought tolerance. However, these studies have either compared two contrasting cultivars [34, 35] or examined the effects of drought on specific tissues within a single cultivar [36–40]. These studies are limited in scope as a comparative transcriptomic analysis of only two cultivars is unlikely to reveal patterns that are characteristic of grapevines as a whole. The diversity of existing grapevine cultivars enables more comprehensive research into the effects of drought on cultivars with varying levels of resilience. However, no such studies have previously been conducted.

Thus, this study aimed to rank 30 grapevine cultivars according to their drought tolerance, based on changes in phenotypic and biochemical parameters. A comparative transcriptomic analysis was conducted on several cultivars with varying levels of drought tolerance to preliminarily identify common patterns and biological processes involved in the response to this type of stress across all cultivars. This approach will help to identify areas for further research into the mechanisms of grapevine drought tolerance.».

 REV: The paper mentions that nine phenotypic traits were examined but does not adequately justify why these specific traits were chosen over others, or how their relevance to drought tolerance was established prior to the study. A more robust explanation of trait selection is needed.

Author: We agree with the comment. We added text to the introduction – «Several mechanisms have been evolved to protect plant from adverse effect of drought stress [16]:

Several reactive oxygen species are generated during drought induced oxidative stress which are cytotoxic in nature. Plant have evolved complex antioxidant defense mechanisms both enzymatic (superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR) etc.) and non-enzymatic (glutathione, alpha-tocopherol, flavonoids, carotenoids, ascorbate etc. [17]) to defend against osmotic stress [18]. Studies showed differences have been observed in activity of antioxidative enzymes and levels of their gene expression in tolerant and sensitive genotypes [19].

Plants adjust osmotic pressure by the production of compatible solutes under salt and drought stress conditions. It includes sucrose, proline, quaternary ammonium compounds (hydroxyproline betaine, proline betaine, glycine betaine etc.) and trehalose [20,21]. Proline plays main role in protection of plants from osmotic stress and is one of the most studied plant compatible solutes [22]. Accumulation of proline in tissues helps in detoxification of enormous ammonia, osmotic adjustment, membrane stabilization, scavenging of free radicals, shielding photosynthetic process, protecting mitochondrial functions, nitrogen and carbon reserve for growth and development after stress resistance [23].

Plants responds against drought stress by having anatomical, morphological and ultrastructure adaptations. Roots play a very important role in acclimating under stress. Adaptive characters for drought and salt stress tolerance include long roots, intense root system and high root density [24]. Thicker root structure absorbs greater amount of water when compared to thinner roots and roots in high number can get in touch with greater water vapors available in soil [25].»

 REV:  The transcriptomic analysis identified only 55 differentially expressed genes (DEGs) when comparing control and stressed groups across five cultivars. This number is remarkably low for a study of this nature and suggests either a very conservative analysis threshold, insufficient sequencing depth, or that the chosen stress duration/intensity was not optimal for capturing a broader transcriptomic response. This significantly limits the depth of molecular insights.

Author: We fully agree with the Reviewer that the 55 DEGs identified does indeed seem an extremely low count. At the same time, the sequencing depth was sufficiently high for transcriptomic data (> 20 million high-quality reads), so the result obtained cannot be attributed to this. As for the selected stress level and duration of exposure, these were chosen such that no critical changes in either phenotypic or biochemical parameters were observed in the drought-tolerant varieties. This was done deliberately to identify precisely those biological processes that begin to change in response to drought, whilst the drought-tolerant varieties cope with the stress. At this point, mechanisms are activated that provide a response to this stress in all varieties, regardless of the variety’s level of drought tolerance. Since the varieties examined differed in their level of tolerance, it can be assumed that such varieties cannot have many identical defence mechanisms; however, it was precisely this analysis that allowed us to identify common biological processes characteristic of varieties with varying levels of tolerance. We have added a more detailed proposed explanation for this small number of DEGs to the discussion: «Only a small number of common DEGs were identified for the five V. vinifera genotypes under drought stress. This could be explained by the fact that several cultivars with different responses to drought and levels of tolerance to this stress were examined. It can therefore be assumed that these cultivars do not share many of the same defence mechanisms. This view is supported by the observation of significant differences in transcriptomic profiles even among cultivars with similar tolerance levels. Therefore, when comparing cultivars with different levels of drought tolerance, too few differentially expressed genes (DEGs) were found to be common to all of them. These genes probably reflect the general patterns of grapevine response to this type of stress in both sensitive, intermediate and drought-tolerant cultivars».

REV: Phenotypic analysis used 1–6% mannitol for one month, while transcriptomic analyses used 2% mannitol for 6–12 hours. These differences in concentration and duration complicate direct comparison and interpretation.

Author: Thank the Reviewer for this comment. The analysis of phenotypic and biochemical parameters was carried out one month later, as the study examined the long-term effects on plants for the purpose of ranking the varieties. Phenotypic changes cannot be observed after 6–12 hours. Proline was measured after 12 hours. The analysis carried out enabled us to select varieties with varying levels of resistance, as well as the degree and duration of stress exposure for transcriptomic analysis. We did not carry out any direct analysis (e.g. correlation analysis) between transcriptomic and phenotypic data, precisely because of the differing durations of exposure.

 REV: The study identifies DEGs but does not include any experimental validation of these findings (e.g., using qPCR). Without validation, the reliability of the transcriptomic data remains questionable, especially given the low number of identified DEGs.

Author: We are grateful to the Reviewer for this remark. We agree that transcriptome data should be validated with qPCR data. Unfortunately, this is not possible. This was a large-scale preliminary study conducted across various varieties, the aim of which was not to identify specific genes, but to pinpoint the biological processes in which changes occur as a result of drought. This will help to identify areas for further research. However, it should be noted that DeSEQ is stringent to detect outliers and excludes genes with extreme read counts by default and the false positive rate for DEG is 0% at adjusted P values less than 0.05 (Anders et al. 2013; Rajkumar et al. 2015). This information has been added to the Materials and Methods section.

REV:  While the paper mentions general patterns under drought stress, such as antioxidant activation and changes in metabolism, the discussion does not sufficiently highlight novel findings or their broader implications for grapevine breeding or crop management.

Author: Thank the Reviewer for this comment. We discussed the data we obtained in greater detail. The novelty of our study lies in the fact that, for the first time, a transcriptomic analysis was carried out on five grape varieties with different responses to and resistance against drought, and common patterns were identified across all the varieties studied. The Discussion section has been expanded. In addition, the biological processes involved in the response to this type of stress have been more clearly systematized: « Under drought conditions, the following were observed in all the varieties studied: (1) activation of antioxidant defence mechanisms; (2) a reduction in the biosynthesis of cell-wall beta-glucan, cellulose and polysaccharides, which is likely to lead to cell damage; (3) an increase in the biosynthesis of monocarboxylic and fatty acids; (4) an intensification of splicing processes; and (5) a reduction in transitional and transmembrane metal transport, accompanied by an increase in intra-Golgi vesicle transport.»

REV: The paper states that ANOVA was performed for all parameters to estimate the significance of stress, cultivar, and their interaction, and means were separated using LSD at p ≤ 0.05. However, specific details about the ANOVA model (e.g., fixed versus random effects, interaction terms) and post-hoc tests are not fully elaborated for all analyses, which could impact the robustness of the phenotypic conclusions.

Author: Thanks to the reviewer for this comment. We have added information about the ANOVA model details to the Materials and Methods section.

REV: While mannitol is a common osmoticum, its effects can differ from actual water deficit. The study does not sufficiently discuss the potential limitations of using mannitol to simulate drought stress and how this might influence the generalizability of the findings to field conditions.

Author: We agree with the comment. We added text to the introduction – «In vitro culture of plant on a medium having selection agents gives chance to choose and regenerate plants with advantageous traits. The selecting agents generally utilized for in vitro drought screening include polyethylene glycol (PEG), mannitol, sorbitol and sucrose [16]. Sugar alcohols such as mannitol and sorbitol have often been used as metabolic inert osmotic in plant cell culture [26-28]. It can simply penetrate plasma membranes and cell walls resulting into increase in osmotic pressure and leads to plasmolysis [19,28,29].  Mannitol has been often used to control the osmotic potential of the nutrient solutions in order to induce water deficit conditions, especially in the root zone [30,31]. Influence of soil heterogeneity, environmental factors and climatic factors may create difficulties in screening at the field level. Therefore, in vitro screening condition is con-sidered to be beneficial over field screening [32]. Though the effect of drought stress only in in vitro screening cannot be done, but preliminary reports can predict the response of stresses. Further validation of response for stress can be done in the field condition.»

Minor Issues:

REV: Typographical Errors and Grammatical Inconsistencies: There are several minor grammatical errors and awkward phrasings throughout the text (e.g., "grapevines generally shows an avoidance strategy" , "After one month of exposure, thirty grapevine genotypes showed different responses to drought". These should be corrected for clarity and professionalism.

Author: Thank to the Reviewer for their careful attention to the text. We have checked and revised the manuscript.

 

REV: Some figures are referenced generally (e.g., "Fig. 1"), while others are very specific (e.g., "Fig. 1b, d and e").

Author: When we refer to a diagram in general, we are discussing all the information presented in the figure. In some cases, the discussion focuses on specific points, in which case we indicate a specific part of the figure.

REV:  Some figures are difficult to read due to small font sizes and crowded information. Ensuring all figures are clear and legible is crucial.

Author: We checked all the figures and improved them.

Reviewer 2 Report

Comments and Suggestions for Authors

The article "Comparative Phenotype and Transcriptome Profiling in Some Grapevine Cultivars in Response to Drought Stress" by Igor Gavrilenko, Ekaterina Vodiasova, Victoria Uppe, Galina Maletich, Artem Yurevich Pronozin, Yuri Plugatar, Sergey Dolgov, and Pavel Khvatkov examines the feasibility of assessing drought tolerance genotypes based on modeling elevated osmotic pressure in vitro culture using mannitol.

The manuscript complies with the journal's requirements and contains the required sections. There are some inaccuracies and incorrect formatting in several sections, as well as spelling errors in the figures. These issues are easily corrected and do not affect the scientific significance of the work, but they should be addressed.
Key issues:
1. The authors did not justify the choice of mannitol as a compelling tool for in vitro drought studies in the introduction. Alternative parameters or calculated data that allow us to evaluate the relationship between mannitol concentration and osmotic pressure are also not provided. They should be calculated and added (for example, we provided such a table in the work: Lazareva, E. M., Baranova, E. N., & Smirnova, E. A. (2017). Reorganization of interphase microtubules in root cells of Medicago sativa L. during acclimation to osmotic and salt stress. Cell and Tissue Biology, 11(4), 324-334.)
2. At the end of the introduction, the aims and objectives or hypothesis of the study should be clearly stated. This should be supplemented and the consistency of the main results and conclusions should be clearly verified. This will enhance the clarity and significance of the paper and highlight the scientific novelty of this study.
3. The authors should check the spelling of the word "mannitol" in the illustrations; it is misspelled everywhere.
4. The authors should describe the specific mannitol they used and, in accordance with the guidelines, indicate the manufacturer. Please note that you most likely used D-mannitol, as other forms are considered toxic, although they do produce an osmotic effect. Please check.
5. Please indicate the plant cultivation temperature and lamp spectrum. Also, include the humidity and CO2 content. If you used cotton plugs, these should also be noted. It is also customary to include the name of the flasks if photographs are not provided. For example, you likely used conical flasks (Erlenmeyer), as round-bottomed flasks would have shown different results.
6. Please provide more detailed information in the figure captions. According to journal guidelines, figures are placed separately with captions and must be completely understandable without reference to the text. For example, you have not indicated the object of study in Figures 4-8 and 10.
7. Please note that grapevine is a genus that includes up to 80 species. The spelling in all parts is incorrect and does not contain a full description of the object or Latin. It should be borne in mind that you probably only dealt with Vitis vinifera sp
I can't know which genotypes you worked with. I'd venture to assume that the genotypes presented include representatives of all three ecogeographic groups: the eastern group (Vitis vinifera convar. orientalis Negr.), the western European group (Vitis vinifera convar. occidentalis Negr.), and the Black Sea coast (Vitis vinifera convar. pontica Negr.). Although it's possible that the collection also included American varieties, of which there are more than 20. I think this clarification should be made both to the materials and methods, as well as to the discussion, since it's clear that drought tolerance is potentially related to the conditions of origin of the species and hybrids.
I believe that this correction requires a careful review of the text and perhaps some changes to the discussion and conclusions.

After these changes are made, this interesting article may be accepted for publication.

Author Response

We thank the Reviewer very much for the careful reading of our manuscript and for providing useful comments.

We revised the manuscript in accordance with the comments of the Reviewer. Our comments and answers are given below.

Key issues:
REV:  The authors did not justify the choice of mannitol as a compelling tool for in vitro drought studies in the introduction. Alternative parameters or calculated data that allow us to evaluate the relationship between mannitol concentration and osmotic pressure are also not provided. They should be calculated and added (for example, we provided such a table in the work: Lazareva, E. M., Baranova, E. N., & Smirnova, E. A. (2017). Reorganization of interphase microtubules in root cells of Medicago sativa L. during acclimation to osmotic and salt stress. Cell and Tissue Biology, 11(4), 324-334.)
Author: We agree with the comment. We added text to the introduction – «In vitro culture of plant on a medium having selection agents gives chance to choose and regenerate plants with advantageous traits. The selecting agents generally utilized for in vitro drought screening include polyethylene glycol (PEG), mannitol, sorbitol and sucrose [16]. Sugar alcohols such as mannitol and sorbitol have often been used as met-abolic inert osmotic in plant cell culture [26-28). It can simply penetrate plasma mem-branes and cell walls resulting into increase in osmotic pressure and leads to plasmolysis [19,28,29].  Mannitol has been often used to control the osmotic potential of the nutrient solutions in order to induce water deficit conditions, especially in the root zone [30,31]. Influence of soil heterogeneity, environmental factors and climatic factors may create difficulties in screening at the field level. Therefore, in vitro screening condition is con-sidered to be beneficial over field screening [32]. Though the effect of drought stress only in in vitro screening cannot be done, but preliminary reports can predict the response of stresses. Further validation of response for stress can be done in the field condition.»

The text has been changed and parameters have been added. The osmotic pressure was calculated according to Lazareva et al [49] and was: 134.2 kPa for 1.0% mannitol, 268.5 kPa for 2.0% mannitol, 402.7 kPa for 3.0% mannitol, 536.95 kPa for 4.0% mannitol, 671.2 kPa for 5.0% mannitol and 805.4 kPa for 6.0% mannitol.

REV:  At the end of the introduction, the aims and objectives or hypothesis of the study should be clearly stated. This should be supplemented and the consistency of the main results and conclusions should be clearly verified. This will enhance the clarity and significance of the paper and highlight the scientific novelty of this study.
Author: We thank the Reviewer for this comment. We rewritten Introduction according to the Reviewer suggestion and added the study aims:  «Thus, this study aimed to rank 30 grapevine cultivars according to their drought tolerance, based on changes in phenotypic and biochemical parameters. A comparative transcriptomic analysis was conducted on several cultivars with varying levels of drought tolerance to preliminarily identify common patterns and biological processes involved in the response to this type of stress across all cultivars. This approach will help to identify areas for further research into the mechanisms of grapevine drought tolerance.»

REV:  The authors should check the spelling of the word "mannitol" in the illustrations; it is misspelled everywhere.
Author: We agree with the comment. The text has been checked and corrected. We now use the term "mannitol".

REV:  The authors should describe the specific mannitol they used and, in accordance with the guidelines, indicate the manufacturer. Please note that you most likely used D-mannitol, as other forms are considered toxic, although they do produce an osmotic effect. Please check.
Author: We agree with the comment. A clarification was added to the text - (D(-)-mannitol, PanReac, ApplyChem, Spain)

REV:  Please indicate the plant cultivation temperature and lamp spectrum. Also, include the humidity and CO2 content. If you used cotton plugs, these should also be noted. It is also customary to include the name of the flasks if photographs are not provided. For example, you likely used conical flasks (Erlenmeyer), as round-bottomed flasks would have shown different results.
Author: We agree with the comment. We don't use cotton plugs. The text has been changed - and cultured at pH 5.7 were maintained under 16h per a day of photoperiod of a tandem pair of fluorescent lamps Philips TL – D 36W/54 – 765 and PHLUORAOSRAML 36W/77 (light intensity of 65 µmol/m2s) at 25±1°C. After 2 months’ cultivation on PG medium from single-node cuttings (without signs of bacterial or fungal infection), shoots developed, which were cut off and placed in culture vessels (jar with a total volume of 500 ml) containing 50 ml agarized PG medium supplemented with 0.05 mg/l NAA, with seven plants per culture vessel.

REV: Please provide more detailed information in the figure captions. According to journal guidelines, figures are placed separately with captions and must be completely understandable without reference to the text. For example, you have not indicated the object of study in Figures 4-8 and 10.
Author: We agree with the comment. A clarification was added more detailed information to the figure captions.

REV:  Please note that grapevine is a genus that includes up to 80 species. The spelling in all parts is incorrect and does not contain a full description of the object or Latin. It should be borne in mind that you probably only dealt with Vitis vinifera sp. I can't know which genotypes you worked with. I'd venture to assume that the genotypes presented include representatives of all three ecogeographic groups: the eastern group (Vitis vinifera convar. orientalis Negr.), the western European group (Vitis vinifera convar. occidentalis Negr.), and the Black Sea coast (Vitis vinifera convar. pontica Negr.). Although it's possible that the collection also included American varieties, of which there are more than 20. I think this clarification should be made both to the materials and methods, as well as to the discussion, since it's clear that drought tolerance is potentially related to the conditions of origin of the species and hybrids.
Author: We agree with the comment. The text has been changed - “To curate a research collection of grapevines in vitro, vines of thirty genotypes [twenty six cultivars of all three ecogeographic groups: Aligote, Bastardo, Sauvignon, Syrah, Rkatsiteli, Cabernet Sauvignon, Malbec, Cabernet Franc, Pinot Blanc, Pinot Gris, Pinot Noir, Chardonnay, Riesling, Chasselas blanc and Garnacha blanca (Vitis vinifera convar. occidentalis Negr.); Muscat Blanc and Muscat Crima (Vitis vinifera convar. orientalis Negr.); Saperavi and Qoqur (Vitis vinifera convar. pontica Negr.); Akademik Avidzba, Veles, Kefesiya Magaracha, Yaltinskiy bessemyannyy, Livia, Podarok Magaracha and Ruta (Vitis vinifera x American Vitis hybrids); three rootstocks: Kober 5BB (Vitis berlandieri × Vitis riparia), Fercal (Vitis berlandieri x Vitis vinifera) and Selection Oppenheim 4 (SO4; Vitis berlandieri × Vitis riparia); one breeding form - Magarach no. TT2 (V. vinifera ‘Talisman’ × V. vinifera ‘Tomaisky’ selected by the All-Russian National Scientific Research Institute of Vine And Winemaking "Magarach")] were collected from field-grown mother vines at the All-Russian National Scientific Research Institute of Vine And Winemaking "Magarach" (lat.: 44.850984′N, long.: 33.650112′E)”.

Reviewer 3 Report

Comments and Suggestions for Authors

This paper can be published in Plants after a major revision. My criticisms are listed below.

 

Major points

 

1. There is no proof that the presented transcriptome analyses are statistically valid. In fact, three biological repeats should be sequenced for each sample to provide statistically significant RNAseq data. As is clear from the Results and Methods sections, this was not done; only one sample was sequenced for each variety, or for each variety treated with mannitol. Therefore, all of the presented transcriptome analysis data and the conclusions based on this analysis are statistically invalid.

 

2. The authors generally ignore the information on changes in the transcriptomes of grapevines in response to drought that is available in the current literature. In fact, tens of papers have been published on this subject. Eight of these papers are briefly mentioned in lines 423–429 of the 'Discussion' section. However, it is absolutely necessary to make a direct comparison of the results presented in this manuscript with the data published by others in order to place this study in the context of the current knowledge base. This is also necessary to clarify whether the presented results are consistent with or contradictory to other data.

 

3. As indicated in line 304, transcriptome sequencing of various grapevine varieties showed that 0.13–3.10% of the reads obtained from individual samples were grapevine virus sequences. I conclude that there was nearly a 30-fold difference in virus load among the analyzed varieties. Therefore, the revised manuscript should clarify whether the observed virus load correlates with (1) the level of drought resistance or (2) the biological characteristics of plants described in sections 2.1 and 2.2. The results of statistically significant correlation analysis should be included in the revised version of the paper.

 

4. According to the currently accepted policies on data availability and transparency, primary transcriptome sequencing data should be deposited in the NCBI SRA archive and made available to readers of the paper. Since this was not done prior to submitting the manuscript, it must be done before submitting the revised version of the paper. The respective BioProject accession number must be provided in the “Data Availability Statement.”

 

Minor points

 

Lines 15-16: This study investigated the impact of drought stress on 30 grapevine genotypes. - It reads that drought stress influences genotype. A rewording is needed.

Line 16: then ranked according to their degree of drought tolerance and – can be omitted

Lines 17-18: abbreviations should be deciphered

Line 18: What is stress threshold? It should be explained.

Line 21: General patterns of what?

Lines 30-34. The first two sentences in Introductions can be omitted.

Lines 50-52: The sentence should be corrected: The content of … is increased in plants to maintain…

Line 52: reactive oxygen scavenging systems (ROS) – ROS stands for reactive oxygen species. Should be corrected.

Line 71: abbreviations should be deciphered

Line 307: mechanisms underlying cultivars – a rewording is needed.

Line 310: only those – can be deleted

Line 311: the same - should be similar

Line 326: differentially expressed genes – the abbreviation DEGs has been already introduced

Line 351: depending on – according to?

Line 412-416: The first paragraph of Discission is not needed and can be deleted.

Line 433: genotypes – should be cultivars

 

Author Response

We thank the Reviewer very much for the careful reading of our manuscript and for providing useful comments.

We revised the manuscript in accordance with the comments of the Reviewer. Our comments and answers are given below.

Major points

REV: There is no proof that the presented transcriptome analyses are statistically valid. In fact, three biological repeats should be sequenced for each sample to provide statistically significant RNAseq data. As is clear from the Results and Methods sections, this was not done; only one sample was sequenced for each variety, or for each variety treated with mannitol. Therefore, all of the presented transcriptome analysis data and the conclusions based on this analysis are statistically invalid.

Author: We fully agree with the reviewer’s comment that, for a statistically valid analysis, the sample size must be at least N=3. This condition is met in our study, as we are comparing all plants under non-stress conditions (control group) with those exposed to 2% mannitol (stress). The aim of the study was to identify DEGs under drought stress in all varieties. Thus, the sample size was N=5.

REV: The authors generally ignore the information on changes in the transcriptomes of grapevines in response to drought that is available in the current literature. In fact, tens of papers have been published on this subject. Eight of these papers are briefly mentioned in lines 423–429 of the 'Discussion' section. However, it is absolutely necessary to make a direct comparison of the results presented in this manuscript with the data published by others in order to place this study in the context of the current knowledge base. This is also necessary to clarify whether the presented results are consistent with or contradictory to other data.

Author: Thank to the Reviewer for this comment. We have added the necessary information and expanded the Introduction and Discussion sections.

REV: As indicated in line 304, transcriptome sequencing of various grapevine varieties showed that 0.13–3.10% of the reads obtained from individual samples were grapevine virus sequences. I conclude that there was nearly a 30-fold difference in virus load among the analyzed varieties. Therefore, the revised manuscript should clarify whether the observed virus load correlates with (1) the level of drought resistance or (2) the biological characteristics of plants described in sections 2.1 and 2.2. The results of statistically significant correlation analysis should be included in the revised version of the paper.

Author: We would like to thank the Reviewer for their careful consideration of our study. We conducted an analysis to investigate the possible influence of viral infection. No such influence was detected. Firstly, all plants were examined and no phenotypic signs of infection were observed, which suggests that the viruses were in a latent phase. Secondly, no correlation was found between the number of reads mapping to viruses and transcriptional profiles. The percentage of reads mapping to the virus database was also independent of drought exposure.

REV: According to the currently accepted policies on data availability and transparency, primary transcriptome sequencing data should be deposited in the NCBI SRA archive and made available to readers of the paper. Since this was not done prior to submitting the manuscript, it must be done before submitting the revised version of the paper. The respective BioProject accession number must be provided in the “Data Availability Statement.”

Author: Thank to the Reviewer for this comment. We deposited all transcriptome data to the NCBI and add this information to the Manuscript.

 REV: Minor points

Lines 15-16: This study investigated the impact of drought stress on 30 grapevine genotypes. - It reads that drought stress influences genotype. A rewording is needed.

Line 16: then ranked according to their degree of drought tolerance and – can be omitted

Lines 17-18: abbreviations should be deciphered

Line 18: What is stress threshold? It should be explained.

Line 21: General patterns of what?

Lines 30-34. The first two sentences in Introductions can be omitted.

Lines 50-52: The sentence should be corrected: The content of … is increased in plants to maintain…

Line 52: reactive oxygen scavenging systems (ROS) – ROS stands for reactive oxygen species. Should be corrected.

Line 71: abbreviations should be deciphered

Line 307: mechanisms underlying cultivars – a rewording is needed.

Line 310: only those – can be deleted

Line 311: the same - should be similar

Line 326: differentially expressed genes – the abbreviation DEGs has been already introduced

Line 351: depending on – according to?

Line 412-416: The first paragraph of Discission is not needed and can be deleted.

Line 433: genotypes – should be cultivars

Author: We thank to the Reviewer for their careful attention to the text. We have checked and revised the manuscript according to these minor points.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

All comments are addressed properly.

Author Response

We thank the Reviewer very much for the careful reading of our manuscript and for providing useful comments.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript's problems have been resolved. The article can be published.

Author Response

We thank the Reviewer very much for the careful reading of our manuscript and for providing useful comments.

Reviewer 3 Report

Comments and Suggestions for Authors

The paper can be accepted for publication in its current form, provided the bulleted points in the Introduction are converted to regular text.

Author Response

We thank the Reviewer very much for the careful reading of our manuscript and for providing useful comments.

We revised the manuscript in accordance with the comments of the Reviewer. All the bulleted points in the Introduction are converted to regular text.