Effect of Drought and Salinity on Water Relations and Photosynthetic Responses of Tamarix elongata and Haloxylon ammodendron in Wutonggou Desert Tourist Area, Northwest China

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Effect of drought and salinity on water relations and photosynthetic responses of a Tamarix and a Haloxylon in Wutonggou desert tourist area, northwest China

Introduction

• The introduction provides a broad background but lacks a precise identification of research gaps. The authors should explicitly state:
• What specific aspect of photosynthesis under combined drought and salinity stress remains controversial?
• How does this study build upon previous findings?
• The statement: "It is still controversial whether photosynthesis is restricted by diffusional or biochemical limitation..." should be supported with references to recent studies.

Methodology: Experimental Design and Statistical Analysis

• The methodology section should include more details on how sample sizes were determined. How many replicates per treatment were used?
• The ANOVA analysis is mentioned, but post-hoc tests (e.g., Tukey’s HSD) are not specified. Clarify how differences between treatments were tested.
• Were the gas exchange measurements conducted on the same leaves for all conditions? If not, how was leaf selection standardized?

Results

Water potential and ion content:

• The results state that elongata showed a 234% decrease in water potential at high salinity, while H. ammodendron decreased by only 60%. What could explain this large difference? Provide a physiological or ecological interpretation.

Photosynthetic limitations:

• The paper discusses both stomatal and mesophyll limitations. However, the mesophyll limitation (Lm) results need better contextualization with previous studies. How do these findings compare to other desert or halophyte species?

Chlorophyll content:

• The results indicate that high salinity reduced chlorophyll content in ammodendron but not in T. elongata. This is unexpected given that H. ammodendron is considered more stress-tolerant. Provide a clearer explanation.

Discussion

• The discussion needs a stronger comparison between elongata and H. ammodendron:
• Why does elongata appear to tolerate moderate salinity better but suffer more at higher salinity?
• How do the observed physiological responses relate to their natural distribution in arid environments?
• The statement: "H. ammodendron adapted quickly to drought and salt stress than T. elongata" needs to be supported with more evidence. Adaptation should be discussed in terms of evolutionary or physiological mechanisms.
• The role of Na+ accumulation and osmotic adjustment is mentioned but not fully explained. How do these findings contribute to broader knowledge about halophyte adaptation?
• Several figures (e.g., Figures 3, 4, and 6) include a large amount of data but lack sufficient labels or clarifications in the captions.
• Figure 1 (soil water content) needs a clearer explanation of why the highest salinity level resulted in higher VSWC.
• In Table 1, the explanation for uppercase and lowercase letters is somewhat unclear. Reword for clarity.

Abstract:

• The sentence "Lower water potential values were measured in H. ammodendron and T. elongata..." is unclear. Specify the stress conditions under which this occurred.
• "It is also suggested that the C4 plant H. ammodendron is higher drought and salt tolerant than the C3 plant T. elongata..." → should be reworded for better readability.

Grammar and Style:

• Avoid repetitive phrases such as "the two studied species" in multiple sections.
• Use more concise wording in some places. Example:
• "The osmotic effect is the primary constraint affecting the plant growth under salinity." → "Osmotic stress is the primary constraint on plant growth under salinity."
• Some figures lack proper axis labels or units (e.g., chlorophyll content).

 

Author Response

Introduction

The introduction provides a broad background but lacks a precise identification of research gaps. The authors should explicitly state:

What specific aspect of photosynthesis under combined drought and salinity stress remains controversial?

Response: [Agree. According to recent studies, whether photosynthesis is mainly restricted by diffusional or biochemical limitation under abiotic stress remains controversial. We state the sentence in introduction section (L65-67).]

How does this study build upon previous findings?

Response: [Thanks. Most studies show that drought and salt stress inhibit plant growth, and the combination of drought and salt stress further reduce plant growth. However, some works found that moderate salinity level with drought stress would give a positive effect on plant growth for some halophytes. Besides, a study shows low salinity does not decrease but improves photosynthetic performance in Panicum antidotale under drought stress. Therefore, we designed this study to quantify the photosynthetic responses of H. ammodendron and T. elongata in response to the combined stress of salinity and drought. The aims of the study are as follows: (1) to correlate water relations and photosynthetic responses with growth, (2) to verify whether moderate salinity level with drought stress would give a positive effect on plant physiological traits, as observed for other halophytes, (3) to distinguish the stomatal (Ls) and mesophyll (Lm) limitations as well as the stomatal and non-stomatal limitations of photosynthesis.]

The statement: "It is still controversial whether photosynthesis is restricted by diffusional or biochemical limitation..." should be supported with references to recent studies.

Response: [Agree. We added recent references in the revised manuscript (L65-67).]

Methodology: Experimental Design and Statistical Analysis

The methodology section should include more details on how sample sizes were determined. How many replicates per treatment were used?

Response: [Agree. We didn’t determine sample sizes in the methodology section. Four separate replicates for each treatment in the study except for the photosynthetic light-response curves and CO₂ response curves.] Thank you for pointing this out. We agree with this comment. Therefore, we determined sample sizes in 2.2. Soil water content, pH and electrical conductivity (L131, L136), 2.4. Sodium and Potassium concentration (L147) and 2.6. Chlorophyll contents and chlorophyll fluorescence (L183) in the revised manuscript.

The ANOVA analysis is mentioned, but post-hoc tests (e.g., Tukey’s HSD) are not specified. Clarify how differences between treatments were tested.

Response: [Agree. We used the Tukey’s HSD test and added post-hoc tests in the statistical analysis section.] Thank you for pointing this out. We agree with this comment. Therefore, we added post-hoc tests in the statistical analysis section (L197-199) in the revised manuscript.

Were the gas exchange measurements conducted on the same leaves for all conditions? If not, how was leaf selection standardized?

Response: [The gas exchange measurements weren’t conducted on the same leaves for all conditions. Fully unfolded young leaves from near the top of the canopy in fully illuminated locations were measured for gas exchange.] Thank you for pointing this out. We agree with this comment. Therefore, we added the leaf selection standardized in the leaf gas exchange section (L155-156) in the revised manuscript.

Results

Water potential and ion content:

The results state that elongata showed a 234% decrease in water potential at high salinity, while H. ammodendron decreased by only 60%. What could explain this large difference? Provide a physiological or ecological interpretation.

Response: [Agree. We provide a physiological interpretation (L363-370) in the revised manuscript.]

Photosynthetic limitations:

The paper discusses both stomatal and mesophyll limitations. However, the mesophyll limitation (Lm) results need better contextualization with previous studies. How do these findings compare to other desert or halophyte species?

Response: [Agree. Unfortunately, it's hard to contextualize the mesophyll limitation (L_m) results with previous studies. Because recent studies use the conductance of carbon dioxide from the substomatal cavities to the initial sites of CO₂ fixation (g_m) to study mesophyll limitations. Our method is different, we can only make qualitative comparison in the discussion section.]

Chlorophyll content:

The results indicate that high salinity reduced chlorophyll content in ammodendron but not in T. elongata. This is unexpected given that H. ammodendron is considered more stress-tolerant. Provide a clearer explanation.

Response: [Thanks. Based on your comment, we believe that there may be some misunderstandings here. In the original manuscript, we mean high salinity reduced chlorophyll content in both two species. Drought also reduced chlorophyll content of the two species, but the effect on chlorophyll content in T. elongata was not significant. We describe “Drought stress led to a significant decrease of Chl content in H. ammodendron but not in T. elongata.” in the original manuscript.]

Discussion

The discussion needs a stronger comparison between elongata and H. ammodendron:

Why does elongata appear to tolerate moderate salinity better but suffer more at higher salinity?

Response: [Thanks. Moderate NaCl (100 mM) induced an obvious increase of Na⁺ accumulation in leaves of T. elongata (figure 4d), which may be beneficial for osmotic adjustment. And we also discussed it in the original manuscript (L336-339).]

How do the observed physiological responses relate to their natural distribution in arid environments?

Response: [Thanks. Plants in arid environment deal with drought stress through various physiological mechanisms, such as reducing water potential and stomatal conductance, increasing water use efficiency. These physiological adaptations enable certain plants to survive and thrive in specific arid environments, thereby affecting their natural distribution patterns. In our study, H. ammodendron had better water relations and more efficient photosynthesis than T. elongata, which contributes to its wide distribution in arid and salinity regions.]

The statement: "H. ammodendron adapted quickly to drought and salt stress than T. elongata" needs to be supported with more evidence. Adaptation should be discussed in terms of evolutionary or physiological mechanisms.

Response: [Thanks. H. ammodendron had better water relations and more efficient photosynthesis than T. elongata, which contributes to its wide distribution in arid and salinity regions. We reinforced this statement in the discussion section (L341-343, L363-370, L443-445).]

The role of Na+ accumulation and osmotic adjustment is mentioned but not fully explained. How do these findings contribute to broader knowledge about halophyte adaptation?

Response: [Thanks. We added some content to broader knowledge about halophyte adaptation (L376-380).]

Several figures (e.g., Figures 3, 4, and 6) include a large amount of data but lack sufficient labels or clarifications in the captions.

Response: [Agree. We supplemented the description of the abbreviations(D, S, and D × S) in the captions of Figures 3(L242-244), Figures 4(L261-263), and Figures 6(L286-288) in the revised manuscript.]

Figure 1 (soil water content) needs a clearer explanation of why the highest salinity level resulted in higher VSWC.

Response: [Agree. We infer that the high salinity treatment severely inhibited the water absorption capacity of the two plants under the same watering conditions, so the soil water content was higher than other salinity treatments.]

In Table 1, the explanation for uppercase and lowercase letters is somewhat unclear. Reword for clarity.

Response: [Agree. We reword to explain uppercase and lowercase letters in Table 1.] Thank you for pointing this out. We agree with this comment. Therefore, we reword the note in Table 1 (L318-319) in the revised manuscript.

Abstract:

The sentence "Lower water potential values were measured in H. ammodendron and T. elongata..." is unclear. Specify the stress conditions under which this occurred.

Response: [Agree. We reword the sentence in Abstract section (L19-21) in the revised manuscript.]

"It is also suggested that the C4 plant H. ammodendron is higher drought and salt tolerant than the C3 plant T. elongata..." → should be reworded for better readability.

Response: [Agree. We reword the sentence in Abstract section (L30-32) in the revised manuscript.]

Grammar and Style:

Avoid repetitive phrases such as "the two studied species" in multiple sections.

Response: [Agree. We used "H. ammodendron and T. elongata" exchanges "the two studied species" in the revised manuscript.]

Use more concise wording in some places. Example:

"The osmotic effect is the primary constraint affecting the plant growth under salinity." → "Osmotic stress is the primary constraint on plant growth under salinity."

Response: [Agree. Modified as suggested.]

Some figures lack proper axis labels or units (e.g., chlorophyll content).

Response: [Agree. Modified as suggested.]

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

I have carefully reviewed your manuscript entitled 'Effect of drought and salinity on water relations and photo-synthetic responses of a Tamarix and a Haloxylon in Wutonggou desert tourist area, northwest China', and while the study presents interesting findings, several key areas require improvement. Below are my detailed comments and suggestions to enhance the quality and clarity of your manuscript:

1. Novelty and Objective:
• Please clearly articulate the novelty of this study at the end of the introduction. What gap in knowledge does this study address?
• Additionally, the manuscript lacks a well-defined objective. Clearly stating the research objectives will provide better direction and coherence to your study.
2. Experimental Setup – Soil Properties:
• Since this is a pot experiment using soil, please specify the type of soil used (e.g., sandy, loamy, clay).
• Provide detailed information on the soil's nutrient composition, particularly nitrogen (N), phosphorus (P), and potassium (K) levels, as these are critical for plant growth.
• Did you assess the sodium chloride (NaCl) content in the soil before using it for the experiment?
• Consider measuring and reporting the Exchangeable Sodium Percentage (ESP), as it is a crucial indicator of soil salinity. Providing this information will strengthen the study’s methodological transparency.
3. Statistical Analysis:
• Did you use a one-way or two-way ANOVA for variance analysis? Please clarify this in the statistical methods section.
• Since two factors (1. Drought and 2. Salinity) are affecting the dependent variables, I strongly recommend using a two-way ANOVA to analyze their interaction effects.
• In your figures, Tukey’s test has been used to indicate significant differences between treatments; however, there is no mention of this test in the statistical analysis section. Please explicitly state that Tukey’s post hoc test was conducted and specify the significance level used (e.g., p < 0.05).
4. Figure 1:
• I recommend converting this figure from a line graph to a bar graph for better visualization of treatment differences. Line graphs are typically used for time-series data, which does not appear to be the case here.
• In the figure caption, please explain the significance of the capital and lowercase letters used in the figure.
• Additionally, an asterisk (*) has been used to denote significant differences. Please clarify the level of significance (e.g., p < 0.05, p < 0.01) in the figure legend.
5. Figure 2:
• Define the abbreviations (D, S, DxS) used in the figure caption.
• There appears to be a discrepancy in the p-value notation: an asterisk (*) is used, which typically represents p < 0.05, but the reported p-value is 0.001. Please resolve this inconsistency.
• I noticed an unusual trend where plant height under drought and 100 mM NaCl is higher than in the control treatment, and there is no significant effect of drought and salinity on plant height under 300 and 500 mM NaCl in Tamarix elongata. Please provide a possible physiological explanation for this unexpected trend.
6. Figure 3:
• The comments from Figures 1 and 2 also apply here.
• Ensure consistency in x-axis and y-axis ticks across all figures. In Figures 1 and 3, the tick marks are placed inside, while in Figure 2, they are outside. Unify this formatting across all figures.
7. Potassium Ion Concentration (Figure 4):
• The observed increase in potassium ion concentration under drought and 500 mM NaCl salt stress is unexpected.
• Based on Figure 1, under drought and salt stress, electrical conductivity (EC) increases, which typically leads to higher electrolyte leakage. This should result in increased sodium ion concentration and reduced potassium retention due to ion displacement.
• Please explain the physiological mechanism behind this increase in potassium ions despite high salinity stress. Does this suggest an active potassium uptake mechanism or stress adaptation response in Tamarix elongata?
8. Figures 7 & 8:
• Convert these figures into bar graphs for better clarity.
• Clearly indicate statistical significance using Tukey’s letters and/or asterisks in the figure.
9. Discussion Section:
• Enhance this section by conducting a more in-depth compare-and-contrast analysis between your findings and previous studies.
• Explain the similarities, as they provide validation for your results.
• Discuss the differences, as they highlight the novelty of your study and potential new insights into plant stress responses.
10. Conclusion Section:

• The conclusion should be revised and strengthened to ensure it is directly supported by the results and discussion sections.
• Clearly summarize the key findings of the study and their broader implications.
• If possible, provide recommendations for future research based on your findings.

Author Response

I have carefully reviewed your manuscript entitled 'Effect of drought and salinity on water relations and photo-synthetic responses of a Tamarix and a Haloxylon in Wutonggou desert tourist area, northwest China', and while the study presents interesting findings, several key areas require improvement. Below are my detailed comments and suggestions to enhance the quality and clarity of your manuscript:

Novelty and Objective:

Please clearly articulate the novelty of this study at the end of the introduction. What gap in knowledge does this study address?

Response: [Agree. Modified as suggested [L101-104].

Additionally, the manuscript lacks a well-defined objective. Clearly stating the research objectives will provide better direction and coherence to your study.

Response: [Agree. We modified the research objectives(L105-109) in the revised manuscript.]

 

Experimental Setup – Soil Properties:

Since this is a pot experiment using soil, please specify the type of soil used (e.g., sandy, loamy, clay).

Response: [Agree. The experiment used sandy soil in Gurbantunggut Desert (L114).]

 

Provide detailed information on the soil's nutrient composition, particularly nitrogen (N), phosphorus (P), and potassium (K) levels, as these are critical for plant growth.

Response: [Agree. The soil nutrient content is low, with nitrogen, phosphorus and potassium about 0.17, 0.37 and 0.19 g· Kg^-1, respectively. We added this sentence(L116-117) in the revised manuscript.]

Did you assess the sodium chloride (NaCl) content in the soil before using it for the experiment?

Response: [We didn’t measure the NaCl content, but we used deionized water wash away the excessive salt from the pot soil before planting.]

 

Consider measuring and reporting the Exchangeable Sodium Percentage (ESP), as it is a crucial indicator of soil salinity. Providing this information will strengthen the study’s methodological transparency.

Response: [Agree. We are sorry for that Exchangeable Sodium Percentage was not measured and we can’t get these measurements because the soil samples have been thrown away.]

 

Statistical Analysis:

Did you use a one-way or two-way ANOVA for variance analysis? Please clarify this in the statistical methods section.

Response: [Agree. We used a two-way ANOVA to analyze interaction effects of drought and salinity treatments. We clarify this in the statistical methods section(L196-197) in the revised manuscript.]

 

Since two factors (1. Drought and 2. Salinity) are affecting the dependent variables, I strongly recommend using a two-way ANOVA to analyze their interaction effects.

Response: [Agree. We did used a two-way ANOVA to analyze interaction effects of drought and salinity. The D × S indicates the interaction of drought and salinity treatments in our figures.]

In your figures, Tukey’s test has been used to indicate significant differences between treatments; however, there is no mention of this test in the statistical analysis section. Please explicitly state that Tukey’s post hoc test was conducted and specify the significance level used (e.g., p < 0.05).

Response: [Agree. Tukey’s post hoc test was conducted and the significance level was specified(L197-199) in the revised manuscript.]

 

Figure 1:

I recommend converting this figure from a line graph to a bar graph for better visualization of treatment differences. Line graphs are typically used for time-series data, which does not appear to be the case here.

Response: [Agree. Figure 1 was converted a bar graph in the revised manuscript.]

 

In the figure caption, please explain the significance of the capital and lowercase letters used in the figure.

Response: [Agree. Because the figure was converted a bar graph, we only used lowercase letters to explain the significance, so we rewrite the significance of the lowercase letters used in the figure (L214-216) in the revised manuscript.]

Additionally, an asterisk (*) has been used to denote significant differences. Please clarify the level of significance (e.g., p < 0.05, p < 0.01) in the figure legend.

Response: [Agree. We clarified the level of significance in the figure legend(L216) in the revised manuscript.]

Figure 2:

Define the abbreviations (D, S, DxS) used in the figure caption.

Response: [Agree. We defined the abbreviations (D, S, D×S) (L227-229) in the revised manuscript.]

There appears to be a discrepancy in the p-value notation: an asterisk (*) is used, which typically represents p < 0.05, but the reported p-value is 0.001. Please resolve this inconsistency.

Response: [Agree. The discrepancy is from two test results. The p<0.001 of drought treatments is result of the two-way ANOVA test, but the asterisk indicates the drought significant difference at same NaCl treatment (Tukey’s post hoc test). ]

I noticed an unusual trend where plant height under drought and 100 mM NaCl is higher than in the control treatment, and there is no significant effect of drought and salinity on plant height under 300 and 500 mM NaCl in Tamarix elongata. Please provide a possible physiological explanation for this unexpected trend.

Response: [Thanks. We provided a physiological explanation (L336-339) in the revised manuscript.]

Figure 3:

The comments from Figures 1 and 2 also apply here.

Response: [Agree. Modified as suggested (L242-244).]

Ensure consistency in x-axis and y-axis ticks across all figures. In Figures 1 and 3, the tick marks are placed inside, while in Figure 2, they are outside. Unify this formatting across all figures.

Response: [Agree. Modified as suggested.]

Potassium Ion Concentration (Figure 4):

The observed increase in potassium ion concentration under drought and 500 mM NaCl salt stress is unexpected.

Based on Figure 1, under drought and salt stress, electrical conductivity (EC) increases, which typically leads to higher electrolyte leakage. This should result in increased sodium ion concentration and reduced potassium retention due to ion displacement.

Please explain the physiological mechanism behind this increase in potassium ions despite high salinity stress. Does this suggest an active potassium uptake mechanism or stress adaptation response in Tamarix elongata?

Response: [Thanks. We explained the physiological mechanism (L396-400) in the revised manuscript.]

Figures 7 & 8:

Convert these figures into bar graphs for better clarity.

Response: [Agree. Modified as suggested.]

Clearly indicate statistical significance using Tukey’s letters and/or asterisks in the figure.

Response: [Agree. Modified as suggested (L311-314).]

Discussion Section:

Enhance this section by conducting a more in-depth compare-and-contrast analysis between your findings and previous studies.

Explain the similarities, as they provide validation for your results.

Discuss the differences, as they highlight the novelty of your study and potential new insights into plant stress responses.

Response: [Thanks. Modified as suggested (L341-343, L363-370, L376-380, L396-400, L443-445.)

Conclusion Section:

The conclusion should be revised and strengthened to ensure it is directly supported by the results and discussion sections.

Clearly summarize the key findings of the study and their broader implications.

If possible, provide recommendations for future research based on your findings.

Response: [Thanks. Modified as suggested (L486-493, L500-501.)

Author Response File: Author Response.doc

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

After reevaluating the revised version of the manuscript. I found significant improvements.