Interactions Between Seasonal Temperature Changes, Activities of Selected Genes and Fruit Quality in Malus domestica Borkh.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I reviewed the manuscript entitled The impact of seasonal temperature fluctuations on genotype x phenotype interactions in Malus domestica Borkh..

In the presented research, the author defines the relationship between local climate changes, fruit parameters, and activity of selected genes with known loci on the reference genome of Malus domestica, which controls fruit development processes.

1. Writing needs to be improved.
2. The title seems unclear, specifically the use of G*P*E. Could you clarify or consider changing to G*E?
3. We primarily use G*E interaction because the genotype * environment changes the phenotypes. Mostly G*E intercation effected the phenotype , i suggest change the whole MS based on G*E interaction
4. According to the material and methods, the author did the qRTPCR, but I have not seen the results. Molecular analyses? which type of analyses the author did and what aout qRTPCR?
5. The author found the phenotypic changes unclear.
6. I suggest the author rewrite the whole manuscript because his/her concept is unclear or does not use good terms.
7. The author selected some genes in the abstract but did not write how to select them. How did you select the specific genes for analysis (AAAA1, AALA1, StG, and AAXA)?
8. Many places needs space , e.g L13
9. L18, FW, IEC, TSS, TA and FF?
10. I think the introduction needs to be short and write based on your title
11. Check L129 cli-mate changes.
12. L141 need space, 135(I), 140(II), 145(III) and 150(IV) after full bloom (DAFB).

Comments on the Quality of English Language

 The English could be improved to more clearly express the research.

Author Response

27.03.2025

Keller-Przybylkowicz Sylwia

The National Institute of Horticultural Science;

Konstytucji 3-go Maja, 96-100 Skierniewice, Poland

Agronomy/MDPI

Dear Managing Editor

Ms. Holly Luan

[Cover Letter]

We would like to thank editorial board and reviewers for every remark that helped us to improve the current version of the manuscript.

We appreciate you and the reviewers for your precious time in reviewing our paper and providing valuable comments.

We have carefully considered the comments and tried our best to address every one of them. We hope the manuscript after careful revisions meet your high standards. The authors welcome further constructive comments if any.

Below we provide the point-by-point responses. All modifications in the manuscript have been highlighted in current version accordingly to the track changes of word document.

Sincerely,

Sylwia Keller-Przybylkowicz,

Sylwia.Keller@inhort.pl

The National Institute of Horticultural Research

Konstytucji 3-go Maja 1/3

96-100 Skierniewice

Poland

Author’s responses to the comments of the reviewer #1 of the manuscript entitled “The impact of seasonal temperature fluctuations on genotype x phenotype interactions in Malus domestica Borkh”, submitted to Agronomy under the tracking number: agronomy-3542555.

PLease do no consider the attached file, it is no the final vaersion 

Thank You for valuable suggestions below;

1. Writing needs to be improved.
2. The title seems unclear, specifically the use of G*P*E. Could you clarify or consider changing to G*E?
3. We primarily use G*E interaction because the genotype * environment changes the phenotypes. Mostly G*E interaction effected the phenotype , i suggest change the whole MS based on G*E interaction

The work has been revised to clarify the hypothesis.

The manuscript is mainly based on temperature changes as the basic environmental factor in the studied evaluation seasons. Considering that the environment affects the phenotype, presented studied focuses in terms of different temperatures in the three evaluated seasons. In addition, the activity of genes of interests that were analyzed also had a significant impact on the phenotype. We also showed that temperatures during the tissue collection period directly affected the activity of selected genes and the fruit evaluated traits, so it seems to us that such a three-factor analysis will allow for a full recognition of the processes occurring in the fruits. Therefore, removing the phenotypic evaluation from the studies will not take into account the full scheme of this mechanism. Table S1 has been added to the supplements file, which explains the temperature range used in the comparative studies. The Figure S1b diagram was additionally presented to indicate the full distribution of monthly temperatures in the three evaluation seasons.

The title has been changed and now sounds like:

An interaction between seasonal temperature changes, activities of selected genes and fruit quality in Malus domestica Borkh.

4. According to the material and methods, the author did the qRTPCR, but I have not seen the results. Molecular analyses? which type of analyses the author did and what about qRTPCR?
5. The author found the phenotypic changes unclear.

The results of the gene expression study (qRT-PCR) were included in the supplements together with the manuscript. We believe that in the corrected version of the manuscript, the influence of temperature changes on gene activity and the studied phenotypic traits of apples is now more clearer. The manuscript has been rearranged according to all reviewer remarks.

All changes were shown in the tracking changes version of the manuscript.

6. I suggest the author rewrite the whole manuscript because his/her concept is unclear or does not use good terms.

The authors accepted the suggestion to improve the work.

The work was properly reedited. All terminologies were checked with the source works of other authors. The authors believe that the work in its current form fully meets the expectations and explains the assumptions that are the subject of the research.

7. The author selected some genes in the abstract but did not write how to select them. How did you select the specific genes for analysis (AAAA1, AALA1, StG, and AAXA)?

Thank You for pointing it out.

Authors admit that indeed, there is a lack of information on the sources of genes. In the final manuscript, we supplemented this data. The Selected gene of Interests (GOI) section and Table 2, where gene loci and their identifiers are given after annotation analysis of balst with the Malus genome was reedited to explain this issue. The following text also includes the full meaning of the role of genes, which is more broadly covered in the discussion.

The new reference positions were added as well:

27. Newcomb RD, Crowhurst RN, Gleave AP, Rikkerink EH, Allan AC, Beuning LL, Bowen JH, Gera E, Ja-mieson KR, Janssen BJ, Laing WA, McArtney S, Nain B, Ross GS, Snowden KC, Souleyre EJ, Walton EF, Yauk YK. Analyses of expressed sequence tags from apple. Plant Physiol. 2006; 141(1):147-66. doi: 10.1104/pp.105.076208.
28. Park S, Sugimoto N, Larson MD, Beaudry R, van Nocker S. Identification of genes with potential roles in apple fruit development and biochemistry through large-scale statistical analysis of expressed sequence tags. Plant Physiol. 2006; 141(3):811-24. doi: 10.1104/pp.106.080994.
29. Many places needs space , e.g L13,
30. L18, FW, IEC, TSS, TA and FF?

Thank you for your both suggestions. Agree, unfortunately, the authors did not avoid such errors. The necessary corrections have been made.

10. I think the introduction needs to be short and write based on your title

Thank You for valuable suggestion.

The manuscript has been improved to meet the suggested requirements. In the revised introduction paragraph, the authors have abandoned the extensive descriptive part concerning fruit development and focused on the hypothesis contained in the title. Now it sounds like:

The seasonal temperature changes have a huge impact on plant yield, fruit development and influence the variability of fruit features accepted by consumers. Global climate change is leading to unusual weather conditions, including temperature fluctuations, as well as annual average temperature increases [1]. In the period of 2000–2020, worldwide temperature have increased approximately about 2 °C and show rising trends in subsequent seasons (source GUS, hhttp://300gospodaka.pl/ (access January 2020) [2]. Many authors explain, that based on the temperature values in particular growing season, it is possible to assess fruit quality, fruit yield, and determine the optimal date for fruit harvesting [3–5]. High temperatures have been known to induce plant heat stress, affecting crucial growth stages, reducing photosynthesis and hampering plant development [6]. Low temperature, especially in apple blooming phase may also result in serious damages in plant organs and reducing plant fruit expansion [7, 8].

Apple (Malus domestica Borkh.) is one of the most economically important fruit tree species grown worldwide. Apple fruits have many benefits for human health and are one of the favorite among consumers [9-11]. Most of them are crunchy, juicy, sweet and rich in vitamins, dietary fiber, polyphenols and minerals [12]. Their quality is determined by a number of individual features, most of which are controlled by many main genes and by smaller polygenes or oligogenes [13-15]. Long-term evolution, gene recombination and natural mutations have resulted in a high level of heterozygosity in the genome of this species [16]. Additionally, targeted selection and the influence of non-additive genetic effects contributed to the increase in the genome variability of apples, expressed in their phenotypic diversity [17].

The average number of days from flowering until harvest as well as temperature conditions seems to be the greatest factors impacting apple phenophases [18, 19]. The methods of calculation of degree-day accumulation or the sum of active temperatures in this precise period are also successfully used to predict the accumulation of desired substances like anthocyanins or sugars [5,20,21]. The phenological phases of fruit development of plants growing in temperate regions strongly depend on temperature conditions especially during winter and spring [22,23], even though, the selection of appropriate onset of temperature calculation is disputable [24]. Fruit organ growth also depends on the temperature accumulation from the flowering to fruit maturity period, and can be stimulated by a fixed number of degree-days which allows comparing different evaluated seasons and locations [4].

The process of apple fruit development is well recognized and includes: the early period, in which intensive cell division and tissue expansion predominate (up to 70 days after full bloom); the maturation stage (lasts from 90 to approximately 150 days after full bloom), when the fruits possesses the prerequisites for use by consumers, and finally the ripening, known as an irreversible stage in which the fruits reach collective maturity [19].

Based on the global microarray analysis Jansen et al. 2008 discovered four major patterns of coordinated gene expression occurred in different fruit developmental stages. First group was represented by genes expressed in floral buds but down-regulated throughout fruit developmental stagres, second group of genes was up-regulated in early fruit developmental stage and down-regulated later, and two additional groups of genes were defined as up-regulated during the middle stages of fruit development and during ripening [19].

This knowledge underlines the complexity of apple fruit development regulated by many genes and their activity may vary according to external (environmental) factors. In this context the temperature fluctuation seems to be the most important in formation of fruit phenotypical attributes [8]  

Thanks to the development of -omics technologies, such as genomics, transcriptomics, proteomics, metabolomics, and phenomics, as well as GWAS (Genome Wide Associated Studies), metabolic GWAS, structural analysis of variability, supported by molecular markers and other molecular technologies, significant progress in genetic research of fruit plants characteristics has been achieved. Despite the long juvenile phase occurring in woody species, such as apple, hampering the application of molecular tools in genotype-phenotype assessment, specific molecular markers (e.g., functional markers) are requested by breeders to improve the marker assisted selection of apples with better fruit quality breeding materials [25].

The presented research aims to fill out the knowledge in understanding the apple fruit ripening mechanism modulated by seasonal temperature changes. Thus explain environment influence also in the activity of selected genes of interest, not characterized so far. We expect the uncovered sequences may contribute in development of functional molecular markers which could be applied for monitoring of fruit ripening process of selected apple cultivars, grown in changing climatic conditions.  

In accordance to the previous remark (6): Explanations of the basis of gene selection have been presented in more detail in the discussion. The MM section has also been supplemented with data on the origin of selected genes, also preceded by analyses of databases. The introduction contains general data on the effect of temperature and explains the significance of its changes on the genetic basis of fruit quality and development, which is of great importance in recognizing the problem, and temperature has been selected as the basic environmental factor.

MM paragraph has been changed. Now it sounds:

For quality and maturity assessments, fruits of four winter apple cultivars: described as early flowering and mid-ripening -,‘Pink Braeburn’ and ‘Pinokio’, early flowering but late ripening ‘Ligol’ and late flowering and late ripening ‘Ligolina’ (both suitable for long-term storage), were collected in three subsequent seasons (2018, 2019 and 2020) and approximately with five-days intervals: 135 (I), 140 (II), 145 (III) and 150 (IV) after full bloom (DAFB). Even though all varieties are considered as winter apples, they significantly differ in flowering time and the time of reaching physiological maturity. The picking time points were chosen according to FB dates for ‘Ligol’ cultivar classified as early flowering (Table 1.).

Fruits were harvested at the same time point intervals for all cultivars, to avoid the potential disequilibrium in their developmental stage. Due to the optimum harvest date (OHD), and for research purpose, the cultivars were classified in groups: early-ripening (‘Ligol’), mid-ripening (‘Pink Braeburn’ and ‘Pinokio’), and late-ripening (‘Ligolina’).

The table 1 caption has been changed:

Table 1. Fruit collection dates.

*FB- date of the full bloom of the apple genotype in evaluated seasons. The date indicated as III represents the commercial fruit harvest date.

11. Check L129 cli-mate changes. Remark has been accepted
12. L141 need space, 135(I), 140(II), 145(III) and 150(IV) after full bloom (DAFB). Remark has been accepted.

The manuscript has been revisited in accordance to English language.

Reviewer 2 Report

Comments and Suggestions for Authors

Comments

The manuscript titled “The impact of seasonal temperature fluctuations on genotype x phenotype interactions in Malus domestica Borkh.” by Keller-Przybylkowicz presents an assessment of selected genes among different apple cultivars over different seasons. The basic idea of the experimental design appears sound. However, the approach used and information provided may not be sufficient. The authors are kindly suggested to address the comments offered below.

Major comments

1. The study selected specific genes for comparison among the selected apple cultivars. However, the reason behind it has not been well explained. A typical approach in a general scenario would have been to select genes based on de novo transcriptome analysis or data assessment of relevant secondary sources. Furthermore, the gene IDs and definitions presented are not informative enough. The authors are suggested to incorporate relevant information, such as the known or expected characteristics of each of the selected genes and the reason behind selecting them, in the manuscript. While doing so, providing valid gene IDs (e.g., MdRKL2), NCBI reference IDs, etc. will greatly enhance the manuscript quality.
2. It is a better alternative to visualize data in plots instead of showing them in tables, as the visualized data is more reader-friendly. Hence, the authors are kindly suggested to make necessary changes. As an alternative, kindly substitute Table 1 with line graphs (with SE if available); Table 4 and Table 5 with either the bar-plot, box-plot, violin-plot, or any other relevant plots; Table 6 would be more informative when combined with heatmap or similar checker-plot. Table 2 can be moved to supplementary. Combined or representative plots from Figure S2 should better be brought to the main content while keeping them as they are in the supplement.
3. Due to the ambiguity of the genes handled in the study, the discussion section appears vague with few corroborating studies. The authors are encouraged to discuss their findings by linking them to the putative function or the gene analyzed in fruit characteristics assessed and comparing them with relevant studies published earlier. The manuscript falls short without making major changes in this regard.

Minor comments

1. Syntactic issues in written English at several places.

Comments on the Quality of English Language

1. The manuscript will greatly benefit from its thorough edit by someone professionally proficient in academic English. The authors are kindly suggested to do so.

Author Response

27.03.2025

Keller-Przybylkowicz Sylwia

The National Institute of Horticultural Science;

Konstytucji 3-go Maja, 96-100 Skierniewice, Poland

Agronomy/MDPI

Dear Managing Editor

Ms. Holly Luan

[Cover Letter]

We would like to thank editorial board and reviewers for every remark that helped us to improve the current version of the manuscript.

We appreciate you and the reviewers for your precious time in reviewing our paper and providing valuable comments.

We have carefully considered the comments and tried our best to address every one of them. We hope the manuscript after careful revisions meet your high standards. The authors welcome further constructive comments if any.

Below we provide the point-by-point responses. All modifications in the manuscript have been highlighted in current version accordingly to the track changes of word document.

Sincerely,

Sylwia Keller-Przybylkowicz,

Sylwia.Keller@inhort.pl

The National Institute of Horticultural Research

Konstytucji 3-go Maja 1/3

96-100 Skierniewice

Poland

Author’s responses to the comments of the reviewer #2 of the manuscript entitled “The impact of seasonal temperature fluctuations on genotype x phenotype interactions in Malus domestica Borkh”, submitted to Agronomy under the tracking number: agronomy-3542555.

Please do not consider the attached file it is not the final vesrion 

Major comments

We would like to thank you very much for your both (1, 3) valuable suggestions.

1. The study selected specific genes for comparison among the selected apple cultivars. However, the reason behind it has not been well explained. A typical approach in a general scenario would have been to select genes based on de novo transcriptome analysis or data assessment of relevant secondary sources. Furthermore, the gene IDs and definitions presented are not informative enough. The authors are suggested to incorporate relevant information, such as the known or expected characteristics of each of the selected genes and the reason behind selecting them, in the manuscript. While doing so, providing valid gene IDs (e.g., MdRKL2), NCBI reference IDs, etc. will greatly enhance the manuscript quality.
2. Due to the ambiguity of the genes handled in the study, the discussion section appears vague with few corroborating studies. The authors are encouraged to discuss their findings by linking them to the putative function or the gene analyzed in fruit characteristics assessed and comparing them with relevant studies published earlier. The manuscript falls short without making major changes in this regard.

The authors have added the literature data to explain the origin of the selected genes in the final version of the work. New reference positions are:

27. Newcomb RD, Crowhurst RN, Gleave AP, Rikkerink EH, Allan AC, Beuning LL, Bowen JH, Gera E, Ja-mieson KR, Janssen BJ, Laing WA, McArtney S, Nain B, Ross GS, Snowden KC, Souleyre EJ, Walton EF, Yauk YK. Analyses of expressed sequence tags from apple. Plant Physiol. 2006; 141(1):147-66. doi: 10.1104/pp.105.076208.
28. Park S, Sugimoto N, Larson MD, Beaudry R, van Nocker S. Identification of genes with potential roles in apple fruit development and biochemistry through large-scale statistical analysis of expressed sequence tags. Plant Physiol. 2006; 141(3):811-24. doi: 10.1104/pp.106.080994.

Additionally, we conducted a balst analysis of selected gene sequences from the NCBI database and assigned their roles based on the most significant match to the annotation database of the Malus genome. In paragraph MM, the literature and in table 2 - gene loci and identificators were introduced based on the library code of Newcomb et al 2006. Gene names are assigned based on published sequence libraries of the ‘Royal Gala’ transcriptome.

In addition, the discussion includes a comparison of our data with the studies of other authors, of which there are relatively few, especially in the aspect of analyses of selected genes. Therefore, it seems to us that the presented manuscript brings new information on the complexity of the regulation of the apple ripening process modulated by seasonal changes in temperature, which is included in the manuscript as the most important and easiest to collect. The newly edited fragment of the discussion sounds like:

 We analyzed an early-expressed group of genes belonging mainly to the class of kinase receptors, which were defined as members of ethylene receptors [27], and their regulation significantly differ depending on the fruits collected few days before fruit harvest. The second group (activated later) of selected genes (AASA and AAYA) were confirmed as essential for sorbitol synthesis in source tissues and its metabolism in sink tissues [27]. Therefore, they can be considered as early functional markers for predicting the ripening stage of apples. However, our data showed that even small seasonal temperature fluctuations (R² ranged from 0.85 to 0.96 - the largest temperature fluctuations were observed in the 2018 and 2019 seasons, and the year-to-year difference was −0.48) can have a significant effect on their activity level. Such an external factors can be involved in gene transportation and their local redirections from different fruit tissues at different stages of their ripening. Our data show a beneficial effect of low temperatures on the activity of genes such as StG, AASA, AALA1, which can prolong the ripening process of ‘Ligol’, ‘Ligolina’, ‘Pinokio’ fruit. The opposite relationship was observed for the 'Pink Braeburn' fruit, for which lower temperature negatively affect the activity of StG, AAA1, AAFB, AALA1, AASA and AAXA genes. This may be due to the inhibition of fruit growth as well as the extension of ripening underlying different genetic processes activated in this apple genotype.

      In our studies, we observed a negative effect of low temperatures on AAFB gene, which could occur due to its initial regulation in the fruit peel, and resulted in the inhibition of its activity (generally observed in ‘Pnokio’ and ‘Ligol’). The positive effect of higher seasonal temperatures on its activity may be caused by the “redirection” of its transcriptional pathway from mesocarp cells to fruit peel cells. Such a mechanism of gene activation/deactivation may be regulated by the presence of transposable regulatory sequences present in the structure of the Malus genome [32-34]. Brown, Telias and colleagues confirmed that genetic variability occurring in the process of fruit development is modulated by such transposable elements [35 -37]. They can affect the alternative transcription start site in gene expression at both the transcriptional and posttranscriptional levels [37].

As it was emphasized by many authors, in the early stage of fruit development, cell cycle genes represented by the group of cyclin dependent kinases CDK [12, 19, 38-40] are activated. In this research we have also observed the group of genes significantly expressed before fruits reach full maturity.  The AAXA, AAAA1 and AABF, were assigned as encoding proteins of cell wall associated kinases receptors. In addition, analyzed AASA gene (encoding enzymes involved in sorbitol metabolism), seem to be crucial later in the process of fruit development. As it was underlined, the accumulated sorbitol and sulfur are quickly metabolized by high activity of SDH (sorbitol dehydrogenase), invertase, SUSY (sucrose synthase), FK (fructokinase) and HK (hexokinase) to demands of plant cell energy, initiating cell division and increase in fruit weight. Then the activity of these enzymes decreases and the starch degradation process is released, resulting in simple sugars accumulation (begins about 90 days after full bloom), and preceding the fruit ripening and determining their sweetness level [41, 42]. Finally, the over-ripening stage, initiated by the initiation of ethylene biosynthesis begins [12, 19, 25].

The expression profiles of these selected genes of interest have not been studied so far, and presented work, sheds light on their usefulness as molecular markers for determining the degree of ripeness of the fruit of four apple varieties.

Thank You for the following suggestions. According to some issues we cannot agree             

2. It is a better alternative to visualize data in plots instead of showing them in tables, as the visualized data is more reader-friendly. Hence, the authors are kindly suggested to make necessary changes.

As an alternative, kindly substitute Table 1 with line graphs (with SE if available)

Table 1 is a table of harvest dates, authors decided to show the data in table plot instead of preparing graph. All 10 fruits of each variety were collected simultaneously at the same time in each period.

Table 4 and Table 5 with either the bar-plot, box-plot, violin-plot, or any other relevant plots; Table 6 would be more informative when combined with heatmap or similar checker-plot. Table 2 can be moved to supplementary.

Thank you for your suggestions.

Such changes in the presented results would be associated with a completely different interpretation and description in the work. The analyses conducted in the work were multifactorial, time-consuming and it seems that many works present their results in the form of correlation tables. Moreover, due to the GrapPadPrism software used, volcano graphs refer to tables 4 and 5, the calculated effect is presented as LogFold change with the appropriate significance. It seems to me that the suggested by reviewer forms of presenting data with graphs will only be able to show differentiation but will not show the statistical effect between the compared factors.

Combined or representative plots from Figure S2 should better be brought to the main content while keeping them as they are in the supplement.

Thank you for your suggestions,

The remark has been accepted. Authors added diagrams for gene expression profiling evaluated for the (III) 145 DAFB, as they relatively present the gene activity changes at commercial harvest date.  

The title has been changed (according to the suggestion of reviewer 1) and now sounds like:

‘An interaction between seasonal temperature changes, activities of selected genes and fruit quality in Malus domestica Borkh.’

The manuscript has been revisited in accordance to English language

Reviewer 3 Report

Comments and Suggestions for Authors

In the submitted manuscript by Sylwia Keller-Przybyłkowicz and colleagues entitled “The impact of seasonal temperature fluctuations on genotype x phenotype interactions in Malus domestica Borkh.’’, the authors studied the interaction of genotype, phenotype and environment in apple fruit growth. The authors found that some genes may be affected by the low temperature. Among the positives of the manuscript are (a) the tables / S. figures have a good presentation, (b) the MM section is explanatory enough. Among the negative aspects of the manuscript are (a) the introduction section; although it is very large, it is not sufficient for genes background (AAFB, AALA1 etc.) that follow in the remaining sections, (b) The discussion section does not contain information/bibliography that supports or explains/interprets the results of this paper, but only re-reports the results reported in the previous section, (c) Several expressive, grammatical and cognitive errors. Overall, the manuscript is not well written. To my opinion, this manuscript is in line with the aims and scope of Agronomy journal. However, there are several issues that should be carefully addressed.

Major and minor comments,
Abstract section

Line 15: Instead of cv., you should write ‘cultivars (cv.)’

The authors should avoid mentioning gene and physiological traits abbreviations such as FW, IEC, TSS, TA, FF and AAAA1, AALA1, StG and AAXA genes

Fruit physiological traits are FW, IEC, TSS, TA, FF, instead of phenology which are the fruit volume, colour, shape etc.

Line 16: The authors mention ‘eight genes related to fruit ripeness’, you should mention these 8 genes and based on what? Bibliography / previous study or something else.

Introduction section

Information about the 8 genes should be reported, how they were found, what they have been associated with, etc.

MM section

Lines 138-139: You mention ‘four winter apple cultivars’, there was no winter / spring / summer / autumn apple cultivars, please rephrase.

The temperature of -1.6 is not explained where it came from, nor are the other temperatures.

You should be stated when the commercial harvest of the apple cultivars was.

internal ethylene concentration (IEC): I am not familiar with this measurement; you should provide more information and some bibliography that has been used. 1ml from the core (which is solid, without internal voids) seems utopian to me. Please reconfirm the method of measuring ethylene production as in most studies the methods of enclosing the fruit in jars are used and either statically or dynamically method is used for the determination of the ethylene production in fruits.

Result section

Line 334: ‘(presented as volcano plots)’, you should mention in supplementary figure 2.

You need to justify why you are studying growing temperatures and earlier temperatures while only focusing on the stages 135 to 150 days after full bloom. I can see any connection, as in addition to temperatures, other environmental and cultivation factors come into play during the development of apple fruits that are not mentioned or studied.

Discussion section

Unfortunately, the discussion section is a repetition of the results, without interpretations and further analyses of the results based on the literature. A complete overhaul is needed in the context of the interpretation of the results of this manuscript.

I cannot understand how figure 2 came about, as there is no mention anywhere of sampling the fruit cortex or peel, only the flesh.

Comments on the Quality of English Language

Line 15: Instead of cv., you should write ‘cultivars (cv.)’

Author Response

Thank You for valuable suggestions and comments.

The authors response are in the attached file

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript titled “The impact of seasonal temperature fluctuations on genotype x phenotype interactions in Malus domestica Borkh.” gives critical information to boost apple fruit production under environmental stress. There are several shortcomings to make the manuscript more fruitful.

Minor remarks:

#Line 188, Table 2: “localization” should be “location”.

#Line 401-402: “higest” should be “highest” throughout the manuscript. Please carefully check for typographical errors throughout the manuscript.

#Line 523, Figure 2: Behind the Gene Source text is difficult to notice because it is literally merged; could you aid in distinguishing the information for better visualization? If possible, modify the figure title to "Schematic circular relationship between gene activity and seasonal temperature fluctuations." Could you please change the symbol to cross (X) for no significant effect? Generally, the "no significant effect" symbol used in the paper represents an inhibition.

Major remarks:

#1: The experiment occurred from 2018 to 2020. Why was not an assessment of the past few years done? Authors should execute experiment for recent years data and correlate it with the previous one.

Author Response

Thank You for valuable suggestions and comments.

The authors response are in the attached file

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author addressed all my comments and suggestions. 

Reviewer 2 Report

Comments and Suggestions for Authors

Comments

The authors have made some changes to the manuscript. However, without track for the changed texts, the manuscript appeared less reviewer-friendly. Nevertheless, the manuscript is relatively better, even though the reviewer still believes that plot visualization would have been better.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript was satisfactorily improved (it fully met my requirements for publication).

Reviewer 4 Report

Comments and Suggestions for Authors

Thank you to the authors for meticulously updating the manuscript.