UVB Stress Induced Changes in Germination and Carbohydrate Mobilization in Chenopodium Quinoa Willd. Seeds
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsChenopodium quinoa Willd. (quinoa) is a very promising crop due to its nutraceutical properties, however, this agricultural crop may be affected by ultraviolet B (UVB) radiation as depletion of stratospheric ozone layer, including seed germination, plant growth and yield, although it was reported that this crop has strong tolerance to extreme conditions including high UVB. Lab experiments were designed to investigate effects of UVB radiation on C. quinoa seed germination and reveal mechanism underlined from changes in amylase enzyme activities and the mobilization of starch reserves. This study is useful and significant in scientific and practical aspects.
The experiments were largely well designed and properly conducted, and useful data and results acquired. After reviewing paper, I have suggestion and comments listed as following:
- Materials and Methods, Figure 1a in Results. I think that it is necessary to tell readers clearly how many seeds (seed number and replicates) were used for germination test.
- Materials and Methods, Figure 1a in Results. No viability assessment was performed before finishing germination test, thus it was unknown whether the ungermination seeds were viable.
- The Y-axis in Figure 4 should be labeled as “Starch content (mg g-1 FW)”
- I assessed neither language nor data analysis because I am not expert at these aspects.
I think this paper can be accepted after the authors respond these comments properly.
Comments on the Quality of English LanguageI assessed neither language nor data analysis because I am not expert at these aspects.
Author Response
1. Materials and Methods, Figure 1a in Results. I think that it is necessary to tell readers clearly how many seeds (seed number and replicates) were used for germination test.
Reply: We have now specified in the Materials and Methods section the details as suggested.
2. Materials and Methods, Figure 1a in Results. No viability assessment was performed before finishing germination test, thus it was unknown whether the ungermination seeds were viable.
Reply: We thank the reviewer for this observation. In our study, no additional viability test was performed on ungerminated seeds. However, after transferring UVB-treated seeds to recovery conditions, germination reached 100% within 48 HAI (Figure 2A), demonstrating that ungerminated seeds during UVB exposure were viable but delayed in germination. This information has been clarified in the Materials and Methods and Results sections.
3. The Y-axis in Figure 4 should be labeled as “Starch content (mg g-1 FW)”
Reply: Corrected as suggested.
4. I assessed neither language nor data analysis because I am not expert at these aspects.
Reply: We appreciate the reviewer’s overall evaluation.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe paper reports experimental results concerning the effect of UVB radiation on germination of quinoa seeds and on some biochemical features of starch consumption during germination.
Here are some specific comments aimed to improve the scientific quality of the article:
Row 25: instead of a- and b-amylase please write α- and β-amylase, to be biochemically correct
Rows 30-31: instead of “ultraviolet light” please write “ultraviolet radiation”, because just like the infrared, the ultraviolet is a range of electromagnetic radiations, but it is not light (light has higher wavelengths and lower energy content than UV radiation)
Row 44: in this paragraph of the Introduction please summarize briefly the current knowledge on how UVB radiation affects plants and on how plants react to this stress when tolerance is expressed
Rows 56-57: please delete the expression “for most seeds”, because for every seed germination begins with rehydration
Rows 62-63: please consider that amylase enzymes are protein molecules, which can be directly damaged by UVB radiation (mainly where aromatic amino acids occur in the molecular structure), beside altering signaling pathways
Row 69: along with the aim of the study, please try to formulate a starting hypothesis for the research or one or two questions to be answered by the planned investigations
Row 71: germination and root elongation are not “morphological data” (because morphology refers to the shape of different parts of the plant body), but rather developmental and growth data
Row 88: there is something wrong with part C of Figure 1, in the second row (UVB treated seeds): how can the radicle be shorter at 12 h then earlier, at 9 h, considering that elongation cannot be reversible?
Rows 116-117: Is there any evidence for the fact that there is sucrose in the quinoa seeds before mobilization of starch reserves begins? Because usually tissues that store starch grains do not simultaneously store sucrose. Please reconsider the affirmation.
Rows 140-141: the formulation “UVB samples showed noticeably light decrease in starch content when compared to their respective control” may be confusing for the readers, because according to Figure 4, the UVB-treated seeds had a higher starch content than their control counterparts; please rephrase the statement to be as clear as possible
Rows 151-153: amylases do not convert starch into glucose, but only into oligosaccharides (dextrins) and eventually maltose molecules are generated; glucose molecules result after the action of maltase enzyme, and conversion of glucose to sucrose is a subsequent process; so amylases by themselves do not produce glucose and sucrose from the starch reserves, please rewrite the affirmation to be scientifically correct and complete
Rows 157-158: please explain briefly how β-amylase activity could be present even in the dry seeds, before germination
Row 205: please correct the name of the variety to ‘Real’, because intraspecific varieties of cultivated plants should be written between apostrophes and should begin with capital letter
Row 207: please write the term “Petri dishes” with capital P, because it originates from the name of a person
Row 236: please give the time and the gravitational force for centrifugation, because methods should be described with sufficient detail to be correctly reproduced by anyone; the same is valid for row 245
Row 244: reference [26] is not the original publication that describes the protocol of photometric starch determination, please indicate the original article
Rows 274-275: before choosing to use ANOVA and Tukey’s test, the normality of data distribution should be analyzed, because the above-mentioned tests can be applied only if there is a normal distribution; if not, then another pair of tests (e.g. Kruskal-Wallis and Mann-Whitney) should be performed instead
A general comment: It is not clear how climatic characteristics of specifically the Andean region, mentioned in the Introduction, are related to the results of the manuscript, which come from laboratory experiments and not from field conditions in the Andes, with a quinoa cultivar which does not originate in that geographic region.
Author Response
Row 25: instead of a- and b-amylase please write α- and β-amylase, to be biochemically correct
Reply: Corrected.
Rows 30-31: instead of “ultraviolet light” please write “ultraviolet radiation”, because just like the infrared, the ultraviolet is a range of electromagnetic radiations, but it is not light (light has higher wavelengths and lower energy content than UV radiation)
Reply: Corrected.
Row 44: in this paragraph of the Introduction please summarize briefly the current knowledge on how UVB radiation affects plants and on how plants react to this stress when tolerance is expressed
Reply: We thank the reviewer for this valuable suggestion. We have revised the Introduction as suggested.
Rows 56-57: please delete the expression “for most seeds”, because for every seed germination begins with rehydration
Reply: Deleted.
Rows 62-63: please consider that amylase enzymes are protein molecules, which can be directly damaged by UVB radiation (mainly where aromatic amino acids occur in the molecular structure), beside altering signaling pathways
Reply: Revised as suggested.
Row 69: along with the aim of the study, please try to formulate a starting hypothesis for the research or one or two questions to be answered by the planned investigations
Reply: We added two guiding research questions to the Introduction, as recommended.
Row 71: germination and root elongation are not “morphological data” (because morphology refers to the shape of different parts of the plant body), but rather developmental and growth data
Reply: Corrected to “developmental and growth data”.
Row 88: there is something wrong with part C of Figure 1, in the second row (UVB treated seeds): how can the radicle be shorter at 12 h then earlier, at 9 h, considering that elongation cannot be reversible?
Reply: We apologize for the oversight. The reviewer is absolutely right. During figure preparation, the 9h and 12h images in part C (UVB-treated seeds) were mistakenly inverted. The figure has been corrected. We appreciate the reviewer’s careful attention to detail.
Rows 116-117: Is there any evidence for the fact that there is sucrose in the quinoa seeds before mobilization of starch reserves begins? Because usually tissues that store starch grains do not simultaneously store sucrose. Please reconsider the affirmation.
Reply: Yes. As reported in reference n. 19, quinoa dry seeds contain sucrose at approximately 3 g per 100 g seeds DW.
Rows 140-141: the formulation “UVB samples showed noticeably light decrease in starch content when compared to their respective control” may be confusing for the readers, because according to Figure 4, the UVB-treated seeds had a higher starch content than their control counterparts; please rephrase the statement to be as clear as possible
Reply: Rephrased as suggested.
Rows 151-153: amylases do not convert starch into glucose, but only into oligosaccharides (dextrins) and eventually maltose molecules are generated; glucose molecules result after the action of maltase enzyme, and conversion of glucose to sucrose is a subsequent process; so amylases by themselves do not produce glucose and sucrose from the starch reserves, please rewrite the affirmation to be scientifically correct and complete
Reply: Corrected.
Rows 157-158: please explain briefly how β-amylase activity could be present even in the dry seeds, before germination
Reply: In other cereals such as barley and wheat, b-amylase is known to be present in dry seeds, accumulated during seeds development. We added an explanation, supported by references (Loreti et al. 1998; Guglielminetti et al. 1995).
Row 205: please correct the name of the variety to ‘Real’, because intraspecific varieties of cultivated plants should be written between apostrophes and should begin with capital letter
Reply: corrected.
Row 207: please write the term “Petri dishes” with capital P, because it originates from the name of a person
Reply: Corrected.
Row 236: please give the time and the gravitational force for centrifugation, because methods should be described with sufficient detail to be correctly reproduced by anyone; the same is valid for row 245
Reply: Corrected.
Row 244: reference [26] is not the original publication that describes the protocol of photometric starch determination, please indicate the original article
Reply: Corrected. The original publication is now cited.
Rows 274-275: before choosing to use ANOVA and Tukey’s test, the normality of data distribution should be analyzed, because the above-mentioned tests can be applied only if there is a normal distribution; if not, then another pair of tests (e.g. Kruskal-Wallis and Mann-Whitney) should be performed instead
Reply: We confirm that normality of data was tested (Shapiro-Wilk) before applying parametric tests. This information has been added to the Statistical Analysis section.
A general comment: It is not clear how climatic characteristics of specifically the Andean region, mentioned in the Introduction, are related to the results of the manuscript, which come from laboratory experiments and not from field conditions in the Andes, with a quinoa cultivar which does not originate in that geographic region.
Reply: We have now clarified in the Introduction that the reference to Andean climatic conditions serves to highlight the natural environment where quinoa evolved under high UVB, while our laboratory study provides a controlled model to investigate the underlying mechanisms of tolerance. We also note that the cultivar ‘Réal’ originates from Bolivia.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript “ UVB Stress Induced Changes in Germination and Carbohydrate Mobilization in Chenopodium quinoa Willd” by Carli et al emphasizes the relevance of the study of the effect of UVB radiation on quinoa seed germination. The introduction is clear and presents enough information to understand the hypothesis.
However, there are some points related to the experimental design that must be improved.
METHODS
In section 3.2 the number of replicates (petri dishes) must be included for the effect of UVB on quinoa germination per biological replicate. This information is also necessary for recovery experiments.
In section 3.3 there is no information about the method used for the quantification of root length. Also, must be included the number of replicates per biological replicates and/or the number of individual roots (n).
RESULTS
Figure 1. In panels A, B and C, the x-axis is confusing. Please change treatment for imbibition time. In the figure legend, indicate the number of replicates and the statistical test used.
Panel C, please improve the quality of these images.
Figure 2. In panel C, please improve the quality of these images.
Figure 3. The x-axis is confusing. Please change treatment for imbibition time in both. In the figure legend, indicate the number of replicates and the statistical test used.
Figure 4. The x-axis is confusing. Please change treatment for imbibition time in both. In the figure legend, indicate the number of replicates and the statistical test used.
Figure 5 and 6. These figures could be improved if you use the model of figure 2 and use two panels; panel A for one graphic and panel B for the images of activity. It will be easier to visualize the comparison between control and UVB treatment. The x-axis is confusing. Please change treatment for imbibition time. In the figure legend, indicate the number of replicates and the statistical test used.
Author Response
METHODS
In section 3.2 the number of replicates (petri dishes) must be included for the effect of UVB on quinoa germination per biological replicate. This information is also necessary for recovery experiments.
Reply: Revised accordingly.
In section 3.3 there is no information about the method used for the quantification of root length. Also, must be included the number of replicates per biological replicates and/or the number of individual roots (n).
Reply: Revised accordingly.
RESULTS
Figure 1. In panels A, B and C, the x-axis is confusing. Please change treatment for imbibition time. In the figure legend, indicate the number of replicates and the statistical test used.
Reply: Corrected as suggested.
Panel C, please improve the quality of these images.
Reply: We regret that higher-quality images are not available. However, we ensured the images are clear enough to distinguish phenotypic differences.
Figure 2. In panel C, please improve the quality of these images.
Reply: As above.
Figure 3. The x-axis is confusing. Please change treatment for imbibition time in both. In the figure legend, indicate the number of replicates and the statistical test used.
Reply: Corrected as suggested.
Figure 4. The x-axis is confusing. Please change treatment for imbibition time in both. In the figure legend, indicate the number of replicates and the statistical test used.
Reply: Corrected as suggested.
Figure 5 and 6. These figures could be improved if you use the model of figure 2 and use two panels; panel A for one graphic and panel B for the images of activity. It will be easier to visualize the comparison between control and UVB treatment.
Reply: We appreciate the suggestion. However, due to the complexity of the data, we believe it is clearer to keep Figures 5 and 6 as separate panels.
The x-axis is confusing. Please change treatment for imbibition time. In the figure legend, indicate the number of replicates and the statistical test used.
Reply: Corrected as suggested.
