The Effects of Low-Temperature Stress on the Physiological Characteristics and Active Components of Ginseng Under Different Soil Moisture Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study presents relevant an important research on the effects of adverse weather conditions in interaction with soil moisture levels on the ginsenoside content of different age ginseng plants. However, the significance of the study does not come across strongly enough.

The manuscript can be improved by highlighting the background and importance of the work more clearly in the introduction. Please see detailed comments in the attached document. 

Adequate parameters have been measured but too much detail is described in the Results section and should be streamlined as not to confuse the reader. The authors should consider to present some of the data differently to emphasize the relevance of the study and show interactions between treatments and measured parameters clearer. Please see detailed comments in the attached document. 

The Discussion requires considerable revision to present the quintessence of the study and importance for ginseng production. Please see detailed comments in the attached document. 

The Conclusion should be shortened and focus put on the main findings and their relevance for ginseng production and how to improve the medicinal value under adverse weather conditions.  Please see detailed comments in the attached document. 

Comments for author File: Comments.pdf

Author Response

Abstract

The manuscript can be improved by highlighting the background and importance of the

work more clearly in the introduction. Please see detailed comments in the attached

document.

1. To investigate the variation patterns of relevant physiological indicators and the

differences in active components in ginseng roots of different ages under low

temperature stress, and to explore the disparities in low temperature responses among

ginseng roots of various growth years. This sentence is incomplete. Please revise or

delete.

Rely：Thank you for the questions raised by the reviewers. The abstract has been revised

in detail, and the revised content is highlighted in red in the manuscript. The revised

content is as follows:To untangle the impact mechanism of low temperature stress on the

physiological indices and accumulation of active components in ginseng of different ages

under varying soil moisture conditions, this study systematically analyzed the

physiological response characteristics and variation patterns of active components in

ginseng of different ages subjected to combined low temperature and water stress. The

aim was to elucidate the adaptation mechanism of ginseng to abiotic stress, providing a

theoretical basis for optimizing ginseng cultivation management practices and enhancing

the quality of medicinal materials.

2. What do the abbreviations behind the moisture levels stand for? Please add

explanation also in Materials & Methods.

Rely：We appreciate the reviewer's comments. Accordingly, we have made modifications

in the Materials and Methods section, with the revised content highlighted in red.

Additionally, in the Abstract, we have introduced "ES, SW, and LQ," which are

abbreviations representing the low soil moisture group, medium soil moisture group,

and high soil moisture group, respectively.

Introduction

The introduction can be improved by adding more relevant literature and by

emphasizing the problem statement more clearly including a hypothesis leading to the

aims of the study.3. There are many types of ginsenosides. Can you please specify which ones are

considered the ones with the most medicinal value.

Rely：We appreciate the reviewer's questions and valuable suggestions. In the article, we

have introduced the potential medicinal value of ginsenosides in the nervous system,

cardiovascular system, immune system, anti-aging, and functional foods, as well as

enumerated the medicinal value of some monomeric ginsenosides. Please refer to

references [5-11] for details. The revised content has been highlighted in red in the article.

4.What is considered optimal soil moisture for ginseng?

Rely：We appreciate the reviewer's comments. The optimal soil moisture for ginseng

growth depends on its growth stage, soil mechanism type, and environmental conditions.

According to research reported by Gao et al. (2019), when the water control mass is

approximately 80% of the soil's saturated water content, it can promote ginseng growth

and saponin synthesis. In our experiment, based on the characteristics of the soil matrix

(peat, vermiculite, soil, and perlite = 1:1:1:0.5), we tested a soil relative water content of

40%-50%. The results indicated that ginseng roots grown under this condition showed no

rot, no atrophy of the root system, solid and plump texture, and well-developed fibrous

roots. Therefore, we consider 40%-50% soil moisture to be suitable for ginseng growth.

5.Please add more references to other relevant research conducted, elaborating also on

the effect of low soil moisture and soil moisture/temperature interaction on plant

responses and neutraceuticals. Has there been similar work done on other medicinal

plants? If yes, please add. If not, you can use that as argument for justifying your own

research.

Rely：We appreciate the reviewer's feedback. In the second paragraph of the introduction

section, we have added relevant content on moisture and moisture-temperature

interactions, citing four references (References 13, 14, 18, 19)。The revised content has

been highlighted in red in the article.

6.Please consider re-phrasing this section, clearing separating and stating the hyposthesis

and aim of the study.

Rely：We appreciate the reviewer's comments. We have revised the research hypothesis

and objectives of the original text, and the modified content is as follows: Previous

studies have indicated that appropriate environmental stress can enhance the stressresistance of plants. This paper focuses on ginseng of different growth years, exploring

the adaptive differences in physiological characteristics and active components of

ginseng under low-temperature stress. Through stress regulation, we aim to improve the

stress resistance and active components of ginseng, providing a theoretical basis for

differentiated water management schemes based on growth duration in the future. The

revised content from lines 87-93 in the text has been highlighted in red.

Materials and methods

7.How many plants per treatment did you use? When was the trial conducted?

Rely: We appreciate the reviewer's comments.In this experiment, 30 pots of ginseng

plants were used for each soil moisture treatment. After the ginseng plants were fully

grown and their leaves had completely unfolded, different soil moisture treatments were

initiated, followed by low-temperature treatment for further experimentation.

8. How was this done? How big was the sample size?

Rely: We appreciate the reviewer's attention to our experimental methods. Following the

completion of the various soil moisture treatments and the full unfolding of the ginseng

leaves, the plants were subjected to low-temperature treatment at 0℃. Root samples were

collected at 4h, 24h, 33h, and 48h of low-temperature exposure. Control plants (0h) from

each moisture group (ES, SW, LQ) that did not undergo low-temperature treatment were

maintained under normal temperature conditions (21±4℃) in a cold light source artificial

climate chamber with a relative air humidity of 40%-60%. The main roots and lateral

roots were carefully excavated, keeping the fibrous roots intact. The samples were

immediately rinsed with distilled water to remove soil from the surface, blotted dry, and

sectioned. They were then placed in liquid nitrogen and stored at -80℃ for physiological

index analysis. Thirty pots of ginseng plants were used for each soil moisture treatment,

and three independent biological replicates were performed at each time point, with

three technical replicates for each determination

9. Please re-phrase. It is not clear what you mean here. What is 'normal' temperature?

21-25℃ as mentioned above? If yes, please add here in brackets.

Rely: We appreciate the reviewer's feedback. In the experimental materials and methods

section, the "normal" temperature refers to 21-25℃, which has been appropriately revised

in the article.10. Please add where these plants were kept and at which soil moisture levels. Did you

also collects root samples from the contoal plants at 0, 4, 24, 33 and 48 hours?

Rely: Thank you to the reviewer for their valuable questions and suggestions. We have

provided additional clarification on the plant storage location and soil moisture control

in the Methods section of the article. The supplementary information is as follows: The

control plants (0h) from each soil moisture water group (ES, SW, LQ) without

low-temperature treatment were stored in a cold light source artificial climate chamber

with a temperature range of 21-25℃ and a relative air humidity of 40%-60%. Root

samples from the control plants were not collected at 0, 4, 24, 33, and 48 hours. Instead,

all control samples were collected uniformly after the completion of soil moisture

treatment.

11. This does not read well. Please consider re-phasing. Do you mean, treatments were

performed in triplicate?

Rely: We appreciate the questions and suggestions raised by the reviewers. The sentence

has been rephrased as follows: Thirty pots of ginseng plants were used for each soil

moisture treatment. Three independent biological replicates were performed at each time

point, and each biological replicate underwent three technical replicate determinations.

12. “The comprehensive cold resistance D value of plants can be classified into three

levels: Level I: D value above 0.7, indicating strong cold resistance; Level II: D value

between 0.5 and 0.7, indicating moderate cold resistance; and Level III: D value below 0.5,

indicating weak cold resistance”. Please provide a reference for this.

Rely: We would like to thank the reviewers for their valuable suggestions. We have

incorporated the relevant reference [24] into the revised statement of this paragraph.

Zhang S, Nimapingcuo, Xu Y, et al. Physiological responses to low temperature stress

and cold tolerance evaluation in three Elymus species[J]. Pratacultural Science, 2016,

33(06):1154-1163.

13. Did you not use repeated measures analysis for the data collected over time?

Which t-test did you use?

Rely: We appreciate the reviewer's questions. Regarding the data analysis and statistical

testing methods used at different time points, our responses are as follows: We indeedconducted a repeated measures analysis three times for data collected at various time

points. We utilized SPSS version 27.0 for data analysis, defining time (0 h, 4 h, 24 h, 33 h,

48 h) as the factor and different soil moisture treatments (low, medium, and high soil

moisture groups) as the dependent variables. The results were averaged from three

independent measurements. A one-way ANOVA combined with Duncan's test was

employed for statistical evaluation of the experimental outcomes. Statistical significance

was considered when P < 0.05. The experimental design of this study involved multiple

group comparisons. The t-test is only suitable for comparisons between two groups,

whereas this experiment required simultaneous analysis of differences among three

groups. One-way ANOVA can test for overall differences among multiple groups in a

single analysis, and when combined with Duncan's test for pairwise comparisons, this

method better suits the multi-group design requirements of our study. Therefore, the

t-test was not used in this research.

Result

Adequate parameters have been measured but too much detail is described in the Results

section and should be streamlined as not to confuse the reader. The authors should

consider to present some of the data differently to emphasize the relevance of the study

and show interactions between treatments and measured parameters clearer. Please see

detailed comments in the attached document.

14. The Results section can be improved by condensing and streamlining the text ,

focusing on the main findings only. A lot of data is presented which can get confusing.

The figures are presented without the control group grown at optimum temperatures

(and 3 soil moisture levels), yet there is reference made to the control group in the text. If

this data was collected it should be presented otherwise no clear conclusion can be

drawn in comparison with the low temperature conditions. If the 0h treatments is meant

to be the control group, it should be more clearly stated.

Rely: We appreciate the detailed suggestions provided by the reviewers regarding the

Results section. In the figures and tables, the 0h group represents ginseng grown under

optimal temperature conditions (21-25℃) and serves as the control group. We have made

corresponding modifications in the Materials and Methods section. The revised content is

as follows: The plants from each soil moisture group (ES, SW, LQ) without

low-temperature treatment were used as controls (0 h) and stored in a cold light source

artificial climate chamber at a temperature of 21-25 ℃ and a relative air humidity of40%-60%.

15. Consider starting this sentence with: In the 2-year-old ginseng group, the MDA ..... to

make it clearer that this section is about the 2-year-old material. Consider doing the same

for the the next section about the 4-year-old material.

Rely: Thanks to the reviewers for their suggestions, which we have made accordingly in

the manuscripts.

16. “ as the duration of low temperature increased” I suggest adding this part at the end

of the sentence for better readability.

Rely: Thanks to the reviewers for their suggestions. We have made corresponding

changes in the manuscripts.

17. What do you mean with control group? The 0h treatment within each soil moisture

group? If yes, please state this clearly in Material &Methods.

Rely: We appreciate the reviewer's question. We have clarified this in the Materials and

Methods section as follows: "The plants from each soil moisture group (ES, SW, LQ)

without low-temperature treatment were used as controls (0 h) and stored in a cold light

source artificial climate chamber at a temperature of 21-25 ℃ and a relative air humidity

of 40%-60%".

18. It might be easier for the reader to follow what is described in the text if treatments

are re-grouped according to moisture levels instead of time. In other words, all sampling

times are displayed within the ES, SW and LQ group. Please consider changing the

graphs.

Rely: We appreciate the valuable suggestions provided by the reviewer regarding the

data presentation. We fully understand the potential benefits of grouping the data

according to humidity levels to enhance readability. However, after careful consideration,

we have decided to retain the current grouping approach based on the following

research requirements: (1) The primary focus of this study is to untangle the relationship

between the duration of low temperature exposure and physiological responses. (2)

Directly demonstrating the threshold changes in physiological indicators as the duration

of low temperature treatment increases.19. Please consider replacing the correlations with principal component analysis as it

should give a clearer picture of how different soil moisture levels affect the physiological

parameters under low temperatures.

Rely: We appreciate the professional advice provided by the reviewer on data analysis

methods. We fully understand the advantages of principal component analysis in

deciphering the interactions of multidimensional data. After careful consideration, we

have made the following improvements: We retained the correlation analysis because the

current study primarily focuses on analyzing the direct relationship between ginseng

subjected to low-temperature treatment and physiological indicators across different soil

moisture groups. Secondly, Pearson correlation analysis can directly quantify the linear

relationship between two indicators. In response to your suggestion, we have conducted

an additional principal component analysis.

20. What about the negative correlations between soluble sugar and proline, and soluble

protein and sucrose (which was also seen in the ES group?)

Rely: Thank you to the reviewer for raising the question regarding the negative

correlation observed between soluble sugars and proline, as well as between soluble

proteins and sucrose in the low and medium soil moisture groups. This negative

correlation may reflect the dynamic trade-off allocation of different osmotic adjustment

substances by ginseng under combined stress conditions.

21. Is this still referring to the control group. If yes, than it contradicts the previous

statement.

Rely: I appreciate the reviewer's inquiry. The control group refers to plants from various

soil moisture groups that were not subjected to low-temperature treatment (0h) and were

stored in a cold light source artificial climate chamber with a temperature range of

21-25℃ and a relative air humidity of 40%-60%. The treatment group consists of plants

that were returned to normal temperature growth after low-temperature exposure until

the harvest period. Corresponding modifications have been made in the Materials and

Methods section.

22. This section is very confusing and it is not clear where to find this data in the

figure/table. Please only focus on the main results otherwise you will loose the reader.

Which are the most important ginsenoside with respect to medicinal value? Maybe you

can focus on these only?Rely: Thank you for the questions raised by the reviewers. We have made detailed

revisions to this section.

conclusion

The Discussion requires considerable revision to present the quintessence of the study

and importance for ginseng production. Please see detailed comments in the attached

document.

23. I think the current sub-headings restrict the discussion on interactions between

temperature and soil moisture levels and their effect on the various parameters measured.

I suggest not using sub-headings or only two sub-headings, one focusing on the

temperature and temperature/soil moisture interaction effect and one focusing on the

plant material age. The role of plant material age on changes in the various parameters

and ginsenosides measured has also not been fully explored. Why does older material

behave differently? Please elaborate. What does all of this mean for ginseng cultivation?

How do plants of different ages need to be treated differently to still ensure highest

ginsenoside content?

Please also add what is still unknown and what future research should focus on.

Rely: Thank you for your careful review and constructive feedback on our study. We

have carefully considered your suggestions and made the following modifications to the

discussion section: Firstly, we have included relevant references to illustrate the

combined effects of temperature and water on various plant parameters. Secondly, we

have adopted your recommendation to optimize the subheadings and have removed the

original ones.

24. The Conclusion should be shortened and focus put on the main findings and their

relevance for ginseng production and how to improve the medicinal value under adverse

weather conditions. Please see detailed comments in the attached document.

Rely: Thank you for your important suggestions on the conclusion. The specific changes

are as follows: This study untangles the response mechanisms of two-year-old and

four-year-old ginseng root systems to low temperature stress under different soil

moisture conditions, as well as the variation patterns of their medicinal components. The

key findings are as follows: (1) In two-year-old ginseng roots, the low/medium soil

moisture groups exhibited the most significant membrane lipid peroxidation damage(highest MDA content) under 48 hours of low temperature exposure. Conversely, the

high soil moisture group delayed membrane damage by increasing SOD activity and

simultaneously enhancing osmotic substances to adapt to the low-temperature

environment. However, this group was unable to maintain the level of osmotic

adjustment substances as the low-temperature stress prolonged. (2) In four-year-old

ginseng roots, the medium soil moisture group maintained the highest SOD activity and

levels of osmotic adjustment substances (soluble proteins, proline). The high soil

moisture group, on the other hand, demonstrated stronger low-temperature tolerance

than the two-year-old plants by continuously accumulating osmotic substances to resist

low-temperature damage. (3) Soil moisture regulation exhibited a dual effect on saponin

metabolism: moderate increases or decreases in soil moisture could promote the

accumulation of total saponins, but low-temperature stress inhibited this process overall.

It is worth noting that the high soil moisture group significantly increased PPT-type

saponin content after low-temperature treatment, providing a new insight for targeted

regulation of medicinal components. This study holds significant implications for

ginseng cultivation management. For four-year-old cultivations, adopting medium soil

moisture management yields the best cold resistance effects. Two-year-old plants should

avoid low soil moisture conditions to prevent exacerbated membrane system damage. In

medicinal production, implementing phased high soil moisture treatment can enhance

the quality of medicinal materials by utilizing its ability to induce PPT-type saponin

accumulation. These discoveries provide a theoretical basis for establishing a precise

water management model based on growth duration and formulating climate-adaptive

cultivation strategies.

25. Please check that the references are formatted according to the author guidelines.

Rely: Thank you for the questions raised by the reviewers. We have checked the format

of the references and made changes according to the author's guide.

The above modifications are marked in red in the manuscript

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Introduction

The objective must be clearly stated. It is stated that the goal is to reduce the impact of low temperatures through humidity management. This assumes a favorable outcome. It is also mentioned that this will result in improved ginseng yield and quality. The objective is very ambitious and cannot be met through research, with the simple fact that yield and quality were not measured. The objective of the study must be reconsidered.

Materials and methods

In 2.1. Add more information about the climate of the experimental site (Koppen climate classification). In example, temperature minimum, average and maximum, relative humidity, radiation and type of soil.  

 

In 2.2. What amount of water provided the moisture levels: Low ES (20%-30%), medium SW (40%-50%), and high LQ (60%-70%).

2.4.2. State the wavelength at which the samples were read. State the retention time of the ginsenosides.

 

Results

3.1. Figures 1a and b are confusing. Are the comparisons between sampling times (0, 4, 24, 33, and 48) or between treatments (ES, SW, LQ)? Please improve the understanding of the figures or provide information in the figure captions that clarify the question.

Same comment in figure 2 a & b.

In section 3.5, the last paragraph should be inserted into the conclusions section.

 

Discussion

Expand the discussion in 4.2. What are the effects of humidity on low temperature resistance?

 

Conclusions are adequate

 

In the attached file, check the form of the references and spaces between words.

Comments for author File: Comments.pdf

Author Response

1. The objective must be clearly stated. It is stated that the goal is to reduce the impact of

low temperatures through humidity management. This assumes a favorable outcome. It

is also mentioned that this will result in improved ginseng yield and quality. The

objective is very ambitious and cannot be met through research, with the simple fact that

yield and quality were not measured. The objective of the study must be reconsidered.

Rely: Thank you for the questions raised by the reviewers. In the manuscripts, we have

made clear the objectives and hypotheses. The specific modifications are as follows in

lines 88-93: This article focuses on ginseng of different growth years, exploring the

adaptive differences in physiological characteristics and active components of ginseng

under low-temperature stress. Regulation through stress can improve ginseng's stress

resistance and active components. This provides a theoretical basis for differentiated

water management strategies based on growth years in the future.

Materials and methods

2. In 2.1. Add more information about the climate of the experimental site (Koppen

climate classification). In example, temperature minimum, average and maximum,

relative humidity, radiation and type of soil.

Rely：We appreciate the reviewer's suggestions. Based on the recommendations, we have

supplemented the information regarding the minimum temperature, average

temperature, maximum temperature, and relative humidity of the experimental site. The

details are as follows: The minimum temperature at the experimental site was 15℃, the

average temperature was 24℃, the maximum temperature was 30℃, and the relative

humidity was 75%. These additions have been highlighted in blue in the article.

3. In 2.2. What amount of water provided the moisture levels: Low ES (20%-30%),

medium SW (40%-50%), and high LQ (60%-70%).

Rely： We appreciate the reviewer's rigorous examination of the experimental details.

Regarding the concern raised about the irrigation water volume data, we provide the

following clarification: The pot experiment is subject to factors such as evaporation and

air humidity, which can lead to variations in irrigation volume. To ensure precise control

over the water status of different soil groups (20%-30%, 40%-50%, 60%-70%), we utilized

a soil moisture detector (QS-SFY-I soil moisture rapid measuring instrument) to monitor

soil moisture levels.4. 2.4.2. State the wavelength at which the samples were read. State the retention time of

the ginsenosides.

Rely：We appreciate the reviewer's rigorous examination of the experimental details.

Our experiment employed ultra-high-performance liquid chromatography coupled with

quadrupole time-of-flight mass spectrometry, which offers advantages in high mass

accuracy and structural resolution capabilities, operating at wavelengths specific to the

UV detector. The retention times for ginsenosides have been included in Table 2.

Results

5. 3.1. Figures 1a and b are confusing. Are the comparisons between sampling times (0, 4,

24, 33, and 48) or between treatments (ES, SW, LQ)? Please improve the understanding of

the figures or provide information in the figure captions that clarify the question.

Rely：We are grateful to the reviewer for their meticulous review of the experimental

specifics. Figures 1, 2, 3, and 4 all present a dual comparison between the differences

among simultaneously reacting treatment groups (ES, SW, LQ) and temporal dynamic

changes (varying durations of stress). The x-axis represents the time gradient of

low-temperature stress (0, 4, 24, 33, and 48 hours), which is used to analyze changes in

physiological indicators within the same treatment group. Annotations have been made

in the figures, highlighted in blue.

6. In section 3.5, the last paragraph should be inserted into the conclusions section.

Rely: We appreciate the reviewer's valuable feedback. Following their suggestions, we

have inserted the relevant content into the conclusion section, specifically from lines 491

to 502.

Discussion

7. Expand the discussion in 4.2. What are the effects of humidity on low temperature

resistance?

Rely: Thank you to the reviewer for their insightful recommendations. In accordance

with their feedback, we have expanded the discussion section to analyze the correlation

between soil moisture and low-temperature tolerance. Additionally, we have included

relevant references to support our findings. Here are the revised details: The combined

effect mechanism of soil moisture and low-temperature tolerance indicates that the

superposition of low temperature and different gradient water stresses can lead to asignificant decrease in soluble protein content, while proline content increases

significantly. This suggests that the combined stress of these two factors is more severe

than water stress alone (Reference 17). Furthermore, under combined stress conditions of

low temperature/15% high humidity and low temperature/3% low humidity, SOD

content increases significantly (Reference 26).

8. In the attached file, check the form of the references and spaces between words.

Rely: Thank you for the questions raised by the reviewers. We have carefully checked

and revised them.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The article submitted by the authors is interesting. The manuscript is written in a logical and orderly manner, but before it is published, the following deficiencies should be corrected:

1. The methodology should precisely state when the ginseng samples were collected for testing.
2. The correlation analysis of physiological indicators in ginseng from different years should be discussed in more detail and, if possible, referred to literature data.
3. I suggest the authors to read the manuscript again carefully in order to eliminate minor language and punctuation errors.

Comments for author File: Comments.pdf

Author Response

1. The methodology should precisely state when the ginseng samples were collected for

testing.

Rely: We appreciate the reviewer's suggestions. Due to the large sample size of the article,

all ginseng samples were collected and stored in liquid nitrogen at -80℃, and later tested

at the same time.

2. The correlation analysis of physiological indicators in ginseng from different years

should be discussed in more detail and, if possible, referred to literature data.

Rely: We appreciate the valuable feedback provided by the reviewers. We fully agree

that analyzing the correlation of physiological indicators among ginseng of different ages

is crucial for elucidating its growth patterns. After systematically searching databases

such as PubMed and Web of Science, we found that most existing studies focus on

comparing physiological indicators within a single age group. The literature on the

physiological indicators or correlations of the same ginseng variety over multiple years is

extremely limited. Therefore, it is difficult to directly cite previous results to support this

part of our analysis. However, in our manuscript, we have added a principal component

analysis of physiological indicators for 2-year-old and 4-year-old ginseng. This analysis

revealed that SOD and proline contribute the most to both 2-year-old and 4-year-old

ginseng.

3. I suggest the authors to read the manuscript again carefully in order to eliminate

minor language and punctuation errors.

Rely: We thank the reviewer for raising these issues. We have carefully reviewed the

article content and corrected the language errors, punctuation, and reference formatting.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for revising the manuscript. Although you have attempted to consider most suggestion, you did not incorporate some of the main suggestions that are important for the reader's understanding. There are also still some inconsistencies regarding treatment codes, which remain confusing.

Unfortunately, the part of the introduction providing the reason for the study was removed in the second manuscript version (i.e. changes in active compound due to adverse weather conditions).

The Results section is still too lengthy, and it is easy to lose the reader with all the detailed information. Focusing on the main results will be beneficial in this regard.

The Discussion also still lacks substance and solid discussion of the results in light of the results and aims of the study and should be revised further.

Some further comments can be viewed in attached document.

Comments for author File: Comments.pdf

Author Response

Thank you for revising the manuscript. Although you have attempted to consider most suggestion, you did not incorporate some of the main suggestions that are important for the reader's understanding. There are also still some inconsistencies regarding treatment codes, which remain confusing. Unfortunately, the part of the introduction providing the reason for the study was removed in the second manuscript version (i.e. changes in active compound due to adverse weather conditions). The Results section is still too lengthy, and it is easy to lose the reader with all the detailed information. Focusing on the main results will be beneficial in this regard. The Discussion also still lacks substance and solid discussion of the results in light of the results and aims of the study and should be revised further. Some further comments can be viewed in attached document.

Rely: We sincerely appreciate your meticulous review and valuable feedback on our manuscript. We have carefully studied all your comments and implemented targeted revisions to the manuscript. Below are our detailed responses and explanations of the specific modifications made：

Abstract

1. There is no need to add this in the abstract, but the percentage moisture, e.g. 30% of the soil saturation capacity, is more important for the reader..My comment in the first version of the manuscript referred to the meaning of the abbreviation of the groups. In other words what does ES or SW or LQ stand for?

Rely: Thank you for the comments provided by the reviewers. In the abstract section, we have rewritten the meanings of abbreviations and added soil moisture content at the end of each group to make it easier for readers to follow. Additionally, in the abstract, we have re-added the content explaining the research rationale, which is as follows: Ginseng growth is susceptible to environmental stress, especially in Northeast China where frequent spring low temperatures and water fluctuations often lead to decreased yields and quality of the medicinal herb. The revised content is highlighted in red.

 

Introduction

2. Response to the feedback regarding "Introduction section lacking research rationale"

Reply: We fully agree with your suggestion that the Introduction section requires a clearer presentation of the research background. In the revised manuscript, we have restored and expanded this section to provide a more comprehensive rationale for the study. Please refer to Lines 78–88, where the modifications are highlighted in red for your convenience.

 

3. Please use italics for all botanical names.

Rely: Thank you for the questions raised by the reviewers. We have made changes in the article. The changes are highlighted in red.

 

4. This is not a scientific term. Please replace with another term and revise the second half of the sentence as it does not sense.

Rely: Thanks to the reviewer for his suggestions, we have revised this sentence as follows：Ginsenoside Rh2 and its aglycone aPPD, as primary metabolites of Panax ginseng, exhibit significant antitumor potential in prostate cancer treatment[10]. Rg2 ameliorates Alzheimer's disease by multi-target regulation of the cerebral metabolic network, alleviating cognitive dysfunction and memory decline[11].。See lines 51-54. The revised content is marked in red。

 

5. “It is noteworthy that an appropriate soil water content not 56 only promotes plant growth but also shields plants from low-temperature damage. 57 When the water content is controlled to approximately 80% of the soil's saturation 58 capacity, both ginseng growth and saponin synthesis are enhanced[12].” This sentence should rather be incorporated in the next section.

Rely: Thanks to the reviewer for your comments. According to your comments, we have added this paragraph to the first sentence of the second paragraph. See lines 55-57, and the revised content has been marked in red.

 

6. “Zhang[15]research” Please replace with: Research conducted by Zhang...。

Rely: Thanks to the reviewers for their comments, we have revised this content, see line 64, and the revised content has been marked in red.

 

7. “Zhang et al[16] studied the roots of Dendrocalamus latiflorus and determined the levels of superoxide dismutase, malondialdehyde (MDA), soluble protein, and soluble sugar content, comprehensively evaluating them through correlation coefficient analysis”. Did these authors also look at soil moisture levels and/or low temperatureconditions? Please add what they found so that the reader can see how it relates to your study.

Rely: Thanks to the reviewers for their comments. The author studied the effects of low temperature stress on Dendrocalamus latiflorus roots. In the manuscripts, we have made corresponding modifications. The modification is as follows：In a study on Dendrocalamus latiflorus roots subjected to 72h low-temperature treatment, research conducted by Zhang [16] found that roots alleviate membrane lipid peroxidation by maintaining high SOD and peroxidase (POD) activity, while reducing plasma membrane permeability through increased unsaturated fatty acid ratios. See lines 66-70. The revised content is marked in red.

 

8. This statement does not contribute to your argument and should rather be deleted.

Rely: Thank you for your meticulous review and valuable feedback. We fully agree with your observation that the highlighted statements lacked sufficient relevance to the core arguments of the study. In response to your suggestion, we have carefully removed those redundant sections to improve the focus and clarity of the manuscript.

 

9. Please consider replacing with the following:

In recent years, abnormal global climate conditions have frequently resulted in low spring temperatures. If soil moisture is insufficient and not timely adjusted through irrigation, ginseng may experience delayed growth and yellowing of leaves due to water deficit following cold spells. Conversely, excessive soil moisture combined with low temperatures can lead to freezing damage, loss of oxygen in the soil, and frozen or rotten roots, thereby affecting the quality and yield of ginseng. Therefore, the aim of the current study was to investigate the effect of soil moisture adjustments under low-temperature stress on changes in physiological characteristics and active components of ginseng of different ages, thereby enhancing ginseng yield and quality and providing a scientific basis for for water management under low temperature conditions caused by climate change.

Rely: Thank you for your thorough review and insightful suggestions. Your proposed enhancements to the manuscript’s wording perfectly address the shortcomings in our original discussion. We are fully aligned with your perspective and have rigorously revised the relevant sections according to your recommended phrasing. The modifications are highlighted in Lines 78–88  of the revised manuscript.

 

Materials and Methods

10. 1 The content is complete

Rely: Thank you for the questions raised by the reviewers. We have fully expressed this sentence as follows：During the trial period, the environmental temperature in the greenhouse was controlled between 15℃ and 30℃ (with an average temperature of 24℃±1℃), and the relative humidity was maintained at 75%±5%。See lines 92-95. The revised content is marked in red.

 

11. “samples” Do you mean pieces?

Rely: Thank you for raising this question. In our study, the term "samples" refers to the cleaned specimens that were cut into small pieces. We have revised this description in the manuscript to ensure clarity. Please refer to Line 120, where the modifications are highlighted in red for your reference.

 

12. Results

Rely: Thank you for your careful review of the manuscript. Feedback on the results section is still too long: We have streamlined the results section, and all the streamlined content has been marked in red. The text font in Figure 5d and Figure 6d has also been adjusted.

 

13. Why are you using different abbreviations here for the different soil moisture groups than above? In table 2 you use CK.

Consider changing it to one consistent code in order not to confuse the reade

Rely: Thank you for your careful review of the details of the manuscript. The inconsistency of abbreviations pointed out by you is very critical, and we have made the modification according to your suggestion. The standard abbreviations of ES (20%-30%), SW (40%-50%) and LQ (60%-70%) are used in the whole text.

 

14. Please provide a better table caption explaining what you mean with LY/CK, LY/SY. According to the text it describes percentage increase. If so, then you should change ginsenoside content in the caption to percentage increase in ginsenosides between soil moisture content groups, or similar.

Rely: Thank you for your valuable comments. According to your guidance, the title of Table 2 has been modified as follows：The fold changes in ginsenoside percentages across varying soil moisture groups under low-temperature stress.

 

Discussion

The discussion still lacks substance, and the discussion of research results and objectives is not in-depth enough, which should be further modified.

Rely: Thanks to the reviewers for their valuable comments. We have made corresponding modifications in the discussion section and added 34 references to echo the high soil water content promoting the accumulation of PPT ginsenoside . The modified content in the discussion section has been marked in red.

 

15. What does this mean here? Are you referring to certain results in Li et al'sresearch? Because it cannot refer to humidity. Please revise.

Rely: Thank you for the questions raised by the reviewers. We have revised reference 25 as follows：Combined cold and high soil moisture stress significantly increased SOD activity in 2-year-old and 4-year-old ginseng roots, while combined cold and low soil moisture stress markedly reduced SOD activity. In 2-year-old ginseng, soluble protein content decreased significantly in both low and high-moisture groups under combined stress, aligning with Li et al.’s findings on the sensitivity of SOD and soluble proteins to cold/high-moisture and cold/low-moisture combined stresses. The humidity of 15% was changed to high-moisture.

 

16. And what about the ES group which had even lower soil moisture?You should discuss both extremes.

Rely: Thanks to the reviewers for their suggestions, we have added the effect of low temperature stress on the accumulation of osmotic regulators in ES group in the manuscript. See lines 397-403.

17. The grammar and other problems pointed out in the manuscript have also been modified accordingly, and the modified content is marked in red.

Thank you for your kind advice and look forward to your further guidance.

Author Response File: Author Response.pdf