Review Reports

Round 1

Reviewer 1 Report

Non-invasive micro-test technology and reciprocal grafting provide direct evidence of contrasting Na⁺ -transport strategies between Cucurbita moschata and Cucurbita maxima

General comments:

This research needs information on two experimental cucurbit species' natural growth and development (including dry weight and photosynthesis). To ensure reproducibility, please provide a clear explanation of the experimental materials and methods, experimental design (including the number of replicates and control), and statistical analysis for each experiment separately in the Materials and Methods section. Additionally, please include references if you adapt or use a published method. Furthermore, there is a data analysis issue that needs addressing. We suggest rephrasing and reanalyzing the data as outlined below.

To make your text easier to read, be sure to include a space between paragraphs and subtitles.

Can you please share the model/s you utilized to evaluate the grafting-combination experiments conducted under salt stress? Also, did you observe any significant interaction? Including these findings in the result section would assist the audience in comprehending the experiment.

Abstract:

The abstract gives a clear summary of the study and its goals, but there are some aspects that could be enhanced.

Starting your abstract with a strong opening sentence that captures the reader's attention is highly recommended for creating a captivating abstract.

It is recommended to avoid using numbered orders when labeling sections such as Introduction, Methods, and etc.

Line 9: The term "previous studies" needs more clarity. Could you please rephrase the sentence to specify the particular focus and achievements of those studies?

Line 14-15: Please provide a clearer statement regarding the "three key genes."

Line 18: In academic writing, it is advisable to use written forms of numbers for figures below ten instead of numerals.

Figure 1. The figure title needs to be revised to clarify that the dry weight measurement was taken for all tissue types.

Introduction:

Line 27-28: Can you tell the location of these 800 million hectares of land?

Line 35-38: Write a sentence that conveys information and includes references.

Materials and Methods:

Line 85: need latitude and longitude for the location.

Line 94: Rephrase the sentence for clarity. Suggestion: Rootstocks were sown 3 days earlier than plants that used for scions.

Line 111: Explain what is the NMT? Add reference for the method.

Line 124-130: Please include the time at which the gas exchange parameters were measured.

Line 132, 256: When conducting scientific experiments, it is essential to specify the exact name/s of the pumpkin species, cultivar, or variety being tested. This allows scientists to draw accurate conclusions based on their findings. Please provide specific names to replace the generic term "two kinds of pumpkin".

Line 133: The leaf pieces seem to vary in size and don't match the sizes mentioned in the Materials and Methods. Can you please explain if the size of the leaf sample affects the leaf color and the chlorophyll fluorescence parameter Fv/Fm? It would be great if you could provide some references for this.

Line 139: Can you please explain the types of planting (grafted or un-grafted) used in gene expression studies?

 

Results:

Make the sub-titles in the Results section easily understandable, consider using short and concise sub-titles that capture the essence of each result. This will enhance clarity and readability for readers.

Line 160: Change the Figure 1 title. Suggestion: … the growth and whole plant dry weight of four grafted combinations, …

Line 168: The phrase "decreased by" lacks clarity and doesn't provide a meaningful comparison. It would be better to rephrase it for clarity.

Line 229-235: Observations should be self-explanatory. “Results showed no remarkable (use appropriate adjective/s suggestions: color and Fv/Fm) differences in the salt tolerance between the two species under 75 mM salt concentration; …”

Line 256: Please rephrase the subheading “Different genotypes of pumpkin … “

 

Discussion:

It is advisable to avoid discussing information that relates to the results.

Line 269: Please provide an explanation of what is meant by the term "previous results."

Line 269-270: I suggest adding a citation to the text.

 

Slight editing is necessary.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

High concentrations of sodium in the soil inhibit plant growth due to the toxic effects of Na ions on photosynthesis and other processes in the leaves. Main plant strategies protecting plant from toxic effects of salinity include limiting their delivery and accumulation in the leaves and sodium sequestration into vacuoles. Present article demonstrates that combination of both mechanisms is most effective. This is achieved by grafting technique based on using Cucurbita moschata as a root stock, which limits sodium delivery to the shoots, and Cucurbita maxima , which is effective in sequestration of sodium into vacuole, - as a scion. The technique can be used in agriculture and article can attract readers. Still I have some remarks, which will be useful for revision of the article.

1.      Lines 31-32. “Excess Na+ in cell interferes with the binding of potassium”. I am not happy with this “binding”. I think “transport” would be better.

2.      Line 62. “the cortex is the main site for storing excess Na+” – I have two remarks concerning this sentence. First, “root” should be added to the “cortex”, since this is a specific root structure. Second, I think another reference should be provided, since I failed to find mentioning “storing excess Na” in the cited article of Munns.

3.      I advise to modify succession of presentation in the figure 3. Since Net Na+ fluxes measured from roots are described first, the figure with this data should be 3A and not 3 C. Fluxes measured from stem should be 3B and leaves should be the last.

4.      I advise to round about most of values in the text. Precision should correspond with the error of the method.

5.      Lines 301-306. “This phenomenon explains why grafting combinations with higher Na+ concentration also have higher K+ concentration (Fig. 2B).” -  It is unclear for me what authors meant. Sodium is generally competing with potassium for transporters and maintaining ion homeostasis under salinity includes opposite effects: a decline in Na accumulation and maintaining K concentration. I advise either to explain clearer what is meant or to delete this passage about potassium.

6.      Legend of Figure 7. “the arrows represent the velocity” – I think it should be mentioned that the size of the arrows (their width) represents velocity

High concentrations of sodium in the soil inhibit plant growth due to the toxic effects of Na ions on photosynthesis and other processes in the leaves. Main plant strategies protecting plant from toxic effects of salinity include limiting their delivery and accumulation in the leaves and sodium sequestration into vacuoles. Present article demonstrates that combination of both mechanisms is most effective. This is achieved by grafting technique based on using Cucurbita moschata as a root stock, which limits sodium delivery to the shoots, and Cucurbita maxima , which is effective in sequestration of sodium into vacuole, - as a scion. The technique can be used in agriculture and article can attract readers. Still I have some remarks, which will be useful for revision of the article.

1.      Lines 31-32. “Excess Na+ in cell interferes with the binding of potassium”. I am not happy with this “binding”. I think “transport” would be better.

2.      Line 62. “the cortex is the main site for storing excess Na+” – I have two remarks concerning this sentence. First, “root” should be added to the “cortex”, since this is a specific root structure. Second, I think another reference should be provided, since I failed to find mentioning “storing excess Na” in the cited article of Munns.

3.      I advise to modify succession of presentation in the figure 3. Since Net Na+ fluxes measured from roots are described first, the figure with this data should be 3A and not 3 C. Fluxes measured from stem should be 3B and leaves should be the last.

4.      I advise to round about most of values in the text. Precision should correspond with the error of the method.

5.      Lines 301-306. “This phenomenon explains why grafting combinations with higher Na+ concentration also have higher K+ concentration (Fig. 2B).” -  It is unclear for me what authors meant. Sodium is generally competing with potassium for transporters and maintaining ion homeostasis under salinity includes opposite effects: a decline in Na accumulation and maintaining K concentration. I advise either to explain clearer what is meant or to delete this passage about potassium.

6.      Legend of Figure 7. “the arrows represent the velocity” – I think it should be mentioned that the size of the arrows (their width) represents velocity

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

The purpose of the study is clearly stated and justified and the experimental results look solid. Yet I have observed several methodological issues (lack of controls) which undermine some principle conclusions. The sequence of presentation can be improved. And, of course, some minor issues are observed.

1. The authors argued that grafting can improve salt tolerance. This conclusion requires a comparison with non-grafted plants. However,  such controls arte present only for the experiments displayed in Fig. 3, but not for those in Figs 1, 2 and 4. Respective controls must be given and a comparison with grafted plants must be discussed.  So far, only a negative impact of MB/JHL grafting and its mechanistic cause (Fig. 7) is supported by the data.

2. Logics. The results on grafted plants are given in Figs 1-4, whereas differential salt tolerance starategies of individula non-grafted plants are presented in Figs 5-6. Should it be better other way around?

3. Relative expression of transporters genes, Fig. 6. Relative to what, control (non salinized) plants? If not, respective controls are needed. If yes, then respective data present fold-changes (up-regulation) by salinity. As far as the changes in different organs are compared, absolute expression in different organs of control plants need to provided, to understand the  importance of particular transporter for a given tissue. 

4. Minor points. First, Cucurbita moschata and C. maxima are different species, not subspicies (line 67). I wonder, whether it is correct to call C. moschata a cucumber, for me both are pumkins, whereas cucumbers belong to a different genus, Cucumis. Further on, I understand that NMT original readings are in pmol cm^-2 s^-1. But cm is not a SI unit. Thus, nmol m^-2 s^-1 appears the correct one. In lines 215 and 217 the correct unit should be s^-1 not S^-1.  Some results are quoted erroneously. e.g. lines 204-206, but there were no significant difference between MB and MB/MB, Fig. 3B. Minor style trimming  is required, e.g. "regionalization" instead of "compartmentalization" on the line 58.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf