Differential Occurrence of Cuticular Wax and Its Role in Leaf Physiological Mechanisms of Three Edible Aroids of Northeast India

Round 1

Reviewer 1 Report

The manuscript "Differential Occurrence of Cuticular Wax and Its Role in Leaf Physiological Mechanisms of Three Edible Aroids of Northeast India" is a study of the cuticular wax (CW) on the leaf epidermis of three edible aroids (Alocasia, Colocasia, and Xanthosoma) and its interaction with different physiological mechanisms and traits. From my perspective there are some minor concerns as follow;

Line 84-88: It is better to combine these two sentences and summarize them

Line 93: please delete the “secondly” word

Line 208- 210: please rewrite theses sentences especially in terms of “22+2 h” and “22 h with hot water treatment for 2 h”.

Line 239: Although Colocasia appears to be different from Xanthosoma in structural, elemental properties and wax microstructures but it is unclear whether they differ, significantly, in wax content or not?

Line 275 and figure 2: Please give some reasons why SFP trait has not changed in Colocasia, with increasing the amount of wax concentration.

Line 356: Why Xanthosoma showed similar l*, a*, b* values in wax and dewax leaves?

• In terms of writing, it is better to revise the whole manuscript including Line 152 (n=8 each), line 174, line 273, etc.
• “Figures” are not clear. Please provide better graphs.
• Are there significant differences between wax and dewax conditions within each aroids, separately, in all traits? It may be interesting to examine the effects of the presence and absence of wax within each plant especially for Chlorophyll content and chlorophyll stability index, RWC, CA, (Pc) infectivity.
• Although the authors provides reasons for the obtained results and well discussed in some traits, but in general, to further confirmation it is necessary to add more references and comparative their results with other closely species. The discussion part is weak from this perspective.
• The analysis of variance and mean comparison tables must be provided in the appendix section.

Author Response

Author’s response to the Reviewer’s comment #1:

Line 84-88: It is better to combine these two sentences and summarize them

Revised as suggested

Line 93: please delete the “secondly” word

The word ‘secondly’ has been deleted as suggested

Line 208- 210: please rewrite theses sentences especially in terms of “22+2 h” and “22 h with hot water treatment for 2 h”.

Revised as suggested

Line 239: Although Colocasia appears to be different from Xanthosoma in structural, elemental properties and wax microstructures but it is unclear whether they differ, significantly, in wax content or not?

CW concentration varied significantly among Alocasia (1.36 mg dm^–2), Colocasia (10.61 mg dm^–2) and Xanthosoma (11.36 mg dm^–2) leaf samples (Fig. 1B). In Fig.1B, the central dotted line in each species depicts the quantity of wax and the left and right dots represent the standard error bars (level of significance at P≤0.01).

Line 275 and figure 2: Please give some reasons why SFP trait has not changed in Colocasia, with increasing the amount of wax concentration.

Colocasia leaf wax possessed lower SPF compared to Alocasia and Xanthosoma due to the smooth, compact, and opaque appearance of the wax microstructure.

Line 356: Why Xanthosoma showed similar l*, a*, b* values in wax and dewax leaves?

Xanthosoma showed similar l^*, a^*, b^* in wax and dewaxed leaves which was an indication of low chlorophyll degradation.

In terms of writing, it is better to revise the whole manuscript including Line 152 (n=8 each), line 174, line 273, etc.

Revised as suggested

“Figures” are not clear. Please provide better graphs.

Figure quality has been improved as suggested. The high resolution (300-500 dpi) images in .tiff format will be provided separately for better visibility.

Are there significant differences between wax and dewax conditions within each aroids, separately, in all traits? It may be interesting to examine the effects of the presence and absence of wax within each plant especially for Chlorophyll content and chlorophyll stability index, RWC, CA, (Pc) infectivity.

Yes. Wax significantly influences the physiological traits among each aroids which has been depicted in the Figures. 1 to Figure.7. 

Although the authors provides reasons for the obtained results and well discussed in some traits, but in general, to further confirmation it is necessary to add more references and comparative their results with other closely species. The discussion part is weak from this perspective.

References on role of wax in the leaf physiological mechanisms aroids/closely related species of Araceae family are limited. The up-to-date references available have been cited in the manuscript. 

The analysis of variance and mean comparison tables must be provided in the appendix section.

The ANOVA/MSS along with the level of significance has been appended as Supplementary Table S1.

Author Response File: Author Response.docx

Reviewer 2 Report

 I suggest improving the quality of the figures and to carry out the small suggestions for the discussion and statistical aspects of figures.

Comments for author File: Comments.pdf

Author Response

Author’s response to the Reviewer’s comment #2:

I have the following suggestions to improve the manuscript:

Abstract

This chapter include the essential aspects

Keywords: change “SPF” for “sun protection factor”

Revised as suggested

Introduction

This chapter correctly covers the essential aspects, it is very well approached.

Line 41-43, update reference

Updated the reference as FAOSTAT 2021.

Line 70: change “cold” by “chilling”

Revised as suggested

The introduction is very well written, includes the fundamental aspects

Thank you very much.

Materials and methods

In equation 1: W_w and L_A, with subscripts

Revised as suggested

Line 174-175: how many measurements were made with the SPAD-502?

Eight measurements each were recorded per treatment (wax and Dewax) each at 0, 15, 30, 60, 120 min. for each species, Aocasia, Colocasia, and Xanthosoma. 

In color, change coordenate l by L.

Revised as suggested

Methods are very well described.

Thank you very much.

In statistical analysis to include test of normality and homogeneity of variances.

The Analysis of variance (ANOVA)/mean sum of square (MSS) along with the level of significance has been appended as Supplementary Table S1 as suggested by the reviewer#1.

Results and discussion

Line 275-278: The discussion must be used to explain their results, otherwise it will remain as a state of the art.

Line 309: include bibliographic reference

Reference [44] included in line no. 309

Lin359-361: improve this discussion because photorespiration is a process that C3 plants generate to avoid photoinhibition and photodegradation of pigments. The pigments are degraded by uncoupling between the photo phase and the C fixation phase, leading to the generation of ROS.

Line 317: cite the figure with these results because they are mentioning that the results were significant (P<0.05).

Video S1 was cited

Figures: Figure 1B: improve legend quality

Image/legend quality has been improved as suggested. The high resolution (300-500 dpi) images in .tiff format will be provided separately for better visibility.

Figure 2. Vertical bars in each column are EE? To include letter of Tukey’s test.

The bars in each column represent the standard error of mean (SEm). Different letters in upper case represent significant difference in SPF among the three aroids and lower case represents significant difference at different wax concentrations according to Tukey’s test.

 

All the figures have been revised with letters of Tukey’s test as suggested

Figure 3. Title of axis “Y” is Contact angle. Changing the color of "Dewas" does not look good.

Revised as suggested.

Figure 4. Bars in each column are EE? To include letter of Tukey’s test.

Revised as suggested.

Figure 5. Improve the quality of the figure, does not look good. Include a comparison test of averages at each time, for example with T-Student. The figure does not look good, but values of “a” must be negative because they indicate green color.

Image quality has been improved as suggested. The high resolution (300-500 dpi) images in .tiff format will be provided separately for better visibility. The graph contains L, a, b value of three aroids at different time points which was little difficult to present in one graph. The dotted lines depict a clear variation among the wax and de waxed leaves. ‘a’ value has been represented as ‘negative’ in the figure.  

Figure 6. Improve the quality of the figure, does not look good. Include a comparison test of averages at each time, for example with T-Student. To include a letter of Tukey's test to compare between species. In axis “y” RWC (%)

Image quality has been improved as suggested. The high resolution (300-500 dpi) images in .tiff format will be provided separately for better visibility.

Different letters in upper case represent significant difference in SPF among the three aroids and lower case represents significant difference at different wax concentrations according to Tukey’s test. The figure has been revised as suggested

Figure S2. Improve image quality.

Image quality has been improved as suggested. The high resolution (300-500 dpi) images in .tiff format will be provided separately for better visibility.

Author Response File: Author Response.docx

Reviewer 3 Report

Specific comments

Title and introduction sections: Review the scope "physiological mechanisms". I believe that the traits studied are not sufficient to generate a scope at the physiological level. The traits studied are more biophysical and biochemical.

Materials and Methods

Line 137-138:  Acronyms were not explained: CSI, RWC, CMI, Pc

Line 189-190: l, a, and b values were transformed to CIE l*, a*, and b* using the algorithms. What are those algorithms?

Line 218: Explain better the variable: Pc infectivity

Line 225: How did you validate the assumptions of the models?. According to the results observed, I consider it necessary to include correlation and regression analyses to analyze the interdependence between the variables studied. 

Lines 226-227: Explain the design better (treatments, replications, experimental units)

Results and Discussion

It is necessary in all figures to indicate the statistical significance obtained with the Tukey test. Also, in all cases, replace (p≤0.05) by the p-value obtained.

Lines 262-263: .. in Alocasia, Colocasia, and Xanthosoma, respectively (data not shown). What data not shown are you referring to? Wouldn't it be more appropriate to include this information in the supplements?

Line 277: ...positively correlated with the protective mechanisms and negatively correlated. What is the r value or significance? The same comment in lines 284, 324

Line 346: ...The greenish leaf color was related to a* values,  which decreased when the time increased. What is the r value and significance? In the figures 5 and 6, regression analyses should support the observed trends 

Line 387: Alocasia leaves showed significantly higher CMI... What is the p-value?

Conclusions

To revise and refine this statement, as more physiological indicators related mainly to leaf gas exchange were missing.: The study results revealed that the leaf cuticular wax coverage 427 in aroids, Colocasia, and Xanthosoma strengthens the leaf epidermis and improves the physiological processes.

The importance of this study in the field of plant breeding was initially mentioned. But this is not elucidated in this section.

 

 

 

Author Response

Author’s response to the Reviewer’s comment #3

Title and introduction sections: Review the scope "physiological mechanisms". I believe that the traits studied are not sufficient to generate a scope at the physiological level. The traits studied are more biophysical and biochemical.

In the present study, we have investigated the role of cuticular wax on leaf physiological properties such as Ch, CSI, moisture loss, RWC, and CMI etc.,

Materials and Methods

Line 137-138:  Acronyms were not explained: CSI, RWC, CMI, Pc

Revised as suggested

Line 189-190: l, a, and b values were transformed to CIE l*, a*, and b* using the algorithms. What are those algorithms?

We have followed the equations as described by Afshari–Jouybari and Farahnaky, 2011.

Line 218: Explain better the variable: Pc infectivity

We have assessed Pc infectivity assay using trypan blue staining to see the early events of infection at 2, 4 and 6 h.

Line 225: How did you validate the assumptions of the models?. According to the results observed, I consider it necessary to include correlation and regression analyses to analyze the interdependence between the variables studied. 

Correlation studies have been included as suggested and updated in the section 2.11

Lines 226-227: Explain the design better (treatments, replications, experimental units)

Revised as suggested

Results and Discussion

It is necessary in all figures to indicate the statistical significance obtained with the Tukey test. Also, in all cases, replace (p≤0.05) by the p-value obtained.

Revised as suggested. We have tested the level of significance at p≤0.01 using ANOVA. The letters obtained from Tukey test have been updated in all the figures

Lines 262-263: .. in Alocasia, Colocasia, and Xanthosoma, respectively (data not shown). What data not shown are you referring to? Wouldn't it be more appropriate to include this information in the supplements?

Revised as ‘Fig. S2’

Line 277: ...positively correlated with the protective mechanisms and negatively correlated. What is the r value or significance? The same comment in lines 284, 324

Correlation table has been incorporated.

Line 346: ...The greenish leaf color was related to a* values,  which decreased when the time increased. What is the r value and significance? In the figures 5 and 6, regression analyses should support the observed trends 

Correlation table has been incorporated.

Line 387: Alocasia leaves showed significantly higher CMI... What is the p-value?

p≤0.01, The letters obtained from Tukey test have been updated in all the figures

Conclusions

To revise and refine this statement, as more physiological indicators related mainly to leaf gas exchange were missing.: 

Influence of wax on leaf gas exchange has not been studied in the present investigation.

The study results revealed that the leaf cuticular wax coverage in aroids, Colocasia, and Xanthosoma strengthens the leaf epidermis and improves the physiological processes. The importance of this study in the field of plant breeding was initially mentioned. But this is not elucidated in this section.

Revised as suggested

 

Author Response File: Author Response.docx