Evaluation of Cadmium Effects on Six Solanum melongena L. Cultivars from the Mediterranean Basin

Round 1

Reviewer 1 Report

The manuscript entitled “Evaluation of Cadmium effects on six Solanum melongena L. cultivars from the Mediterranean basin” describes a study that investigates hydroponically the effects of different Cd concentrations on growth, survival, translocation and physiology of different cultivars of S. melongena. The study is well designed and the ms is well-written.

I believe that this study provides important information on the response of the eggplant cultivars on Cd pollution, but first the authors should provide some additional info (which should be easily provided from the data they already have).

Comments

line 35: The authors report that they investigate Cd tolerance, among other things, but they never provided a tolerance measurement. An easy Cd tolerance index could be the the ratio of root or shoot length of plantlets grown on CdSO4 solutions to the root or shoot lengths of plantlets grown on the control solution (or a variation of that).

line 59: Erase “on”

line 88: The authors should elaborate a bit more on what is the practical purpose of pollution safe cultivars? Are we going to grow eggplants on Cd-polluted areas? In case of pollution, we won't need to throw away the harvest? Please give the practical view of your experiment/results.

line 103: Is the selected metal gradient simulating real nature concentrations? Which concentration corresponds to a natural system, which concentration might be closer to a volcanic soil and which to polluted one? Considering that we are interested in the responses of the cultivars in growing in nature, in volcanic and/or polluted soils, the authors should explain why they used this specific Cd gradient and not another one (e.g wider).

line 118: How did the authors calculate RGR? Biomass divided by 15 days to get mg/day? Usually RGR calculation several consecutive measurements along time (because growth is not necessarily linear – especially in metal toxicity situations).

line 147: The authors should briefly describe the protocol if possible, so that the reader will not have to read another paper.

Figure 1: Please denote significant differentiation between treatment by providing different letters. Please do the same for all figures with comparisons between subjects (e.g. with post-hocs).

Author Response

Dear Reviewer,

herewith we submit the revised version of our manuscript by Filippone et al. We found your comments very helpful for improving the manuscript and included most of them in the present version. We outlined in detail how we addressed all your comments. We hope that this revised manuscript will meet your support and look forward to hearing from you.

Sincerely,

Giovanni Scopece

Department of Biology,

University Federico II of Naples,

Complesso Universitario MSA,

Via Cinthia, I-80126,

Naples, Italy

The manuscript entitled “Evaluation of Cadmium effects on six Solanum melongena L. cultivars from the Mediterranean basin” describes a study that investigates hydroponically the effects of different Cd concentrations on growth, survival, translocation and physiology of different cultivars of S. melongena. The study is well designed and the ms is well-written.

I believe that this study provides important information on the response of the eggplant cultivars on Cd pollution, but first the authors should provide some additional info (which should be easily provided from the data they already have).

Comments

line 35: The authors report that they investigate Cd tolerance, among other things, but they never provided a tolerance measurement. An easy Cd tolerance index could be the ratio of root or shoot length of plantlets grown on CdSO₄ solutions to the root or shoot lengths of plantlets grown on the control solution (or a variation of that).

RESPONSE: We thank the reviewer for this comment and , we now estimate a tolerance index. as the ratio between root length of treated plants and root length of plants grown in control solution (Hakmaoui et al. 2007).

Line 59: Erase “on”

RESPONSE: Done, as suggested

Line 88: The authors should elaborate a bit more on what is the practical purpose of pollution safe cultivars? Are we going to grow eggplants on Cd-polluted areas? In case of pollution, we won't need to throw away the harvest? Please give the practical view of your experiment/results.

RESPONSE: As we explain in the text, our main aim was to identify pollution-safe cultivars to be used for food safety. Our experimental design impedes us to detect Cd in the edible part of the plants, e.g. fruits, but allows to identify cultivars with low translocation efficiency to aboveground organs and to evaluate the levels of expression of genes involved in Cd accumulation. Although this is not a direct assessment of Cd content in organs of the plant generally used for food consumption, leaves from young plants generally have a similar strength of fruits or seeds in terms of sink-source relationship. Accordingly, Shen et al. (2021) compared 30 eggplant cultivars and showed that in high  and low Cd  accumulating cultivars Cd content in fruit was lower than Cd content in leaves. This suggests that Cd content in leaves from young plants might be used for a rapid screening of different cultivars. We added a brief statement in the revised discussions to acknowledge this point. In the conclusions we already mention the need of testing fruits in the more tolerant cultivars to further confirm the screening.

line 103: Is the selected metal gradient simulating real nature concentrations? Which concentration corresponds to a natural system, which concentration might be closer to a volcanic soil and which to polluted one? Considering that we are interested in the responses of the cultivars in growing in nature, in volcanic and/or polluted soils, the authors should explain why they used this specific Cd gradient and not another one (e.g wider).

RESPONSE: Concentration of Cd in Volcanic areas and in contaminated sites is extremely variable and it is difficult to define thresholds that can delimit them or take them apart. However, a soil is generally considered to have a high concentration of Cd when its value is above 8 mg kg^-1 (e.g. Liu et al. 2013). If we convert our concentration values, in our study, we used a wide gradient ranging between the control where Cd is present in traces and 20.8 mg kg^-1 (with four intermediate concentrations: 1.04, 2.08, 5.21, 10.4) to check plant performances even in very highly contaminated soils.

line 118: How did the authors calculate RGR? Biomass divided by 15 days to get mg/day? Usually RGR calculation several consecutive measurements along time (because growth is not necessarily linear – especially in metal toxicity situations).

RESPONSE: We agree on the reviewer’s comment. As we don’t have consecutive measurements along time, in the revised version we renamed the index as “growth index” to avoid confusion with classical RGR index. Growth index was calculated using the following formula: (fresh weight T₁ - fresh weight T₀)/fresh weight T₀.

line 147: The authors should briefly describe the protocol if possible, so that the reader will not have to read another paper.

RESPONSE: Done, as suggested. We added the following description in the material and methods:

We followed the procedure described in [38]: 50 mg of plant material were finely ground and then 1 volume of 80 % (v/v) acetone solution was added. After centrifugation for 5’ at 14000 rpm and 4°C the absorbance was determined by spectrophotometric analysis (Perkin Elmer UV/Vis Spectrophotometer Lambda 25) at the following wavelengths: 470, 646.6, 646.8, 663.2 and 720 nm. The content of chlorophyll a (Chl-a), chlorophyll b (Chl-b), total chlorophyll (Chl-tot) and carotenoid (Car) was quantified by the following equations:

Chl-a = (12.25 x Abs663.2nm) - (2.79 x Abs646.8nm)

Chl-b = (21.50 x Abs646.8nm) - (5.10 x Abs663.2nm)

Chl-tot = (7.15 x Abs663.2nm) + (18.71 x Abs646.8nm)

Car = [(1000 x Abs470nm) - (1.82 x Chl-a) - (85.02 x Chl-b)]/198

Figure 1: Please denote significant differentiation between treatment by providing different letters. Please do the same for all figures with comparisons between subjects (e.g. with post-hocs).

RESPONSE: According to Reviewer’s comment, we added different letters to denote significant differences in figure 1 for the left panel. For the right panel, we reported the results of statistical analyses in the text to avoid confusion on the graph and provide a clearer explanation.

We also added letters in figure 3. In figure 4 and 5 the comparison is pairwise and thus we left stars.

Reviewer 2 Report

Manuscript No.: agriculture-1782472

The manuscript agriculture-1782472 “Evaluation of Cadmium effects on six Solanum melongena L. 2 cultivars from the Mediterranean basin” try to identify pollution-safe cultivars (eggplant) with low translocation efficiency to aboveground organs to be used for food safety. The topic of this manuscript is meaningful and suitable for Agriculture Journal. However, this paper still need revision before accepted.

The critical points are reported below:

1.     In the section of Introduction, Line 70-line 82, there are many papers research on screening for low-translocation efficiency (low accumulation) on agricultural plants. Please check the related studies on eggplant, if have, please added the introduction and references in this paragraph.

2.     In the section of Materials and Methods, Line 116, why choose “15 days”, In my opinion, 15 days is just the growth period in eggplant. People eat the fruit of eggplant, harvest the eggplant when the fruit was growing will be more practical significance and evaluation value? Please explain it.

3.     In the section of Materials and Methods, Line 131-142, the translocation factor and bio-concentration factor are most important index for evaluating heavy metal-tolerant plant. But in your equations, the bioaccumulation coefficient and translocation coefficients seem wrong. Please check the equations, and this article is for your reference: https://doi.org/10.1016/j.chemosphere.2021.130130

4.     In the section of Materials and Methods, 2.8 statistical analysis. Please state n for Germination, Hydroponic culture, and qPCR.

5.     In the section of Results, Line 248, “Cd 5 μM”? It should be “5 μM Cd”, please check across the whole manuscript.

6.     In the section of Results, Line 251, change mg Kg^-1 to mg kg^-1. And check across the whole manuscript.

7.     Figure 5, why some “※” was in the bar graph, some are out of bar graph? Please explain.

8.     In the section of Conclusions. The novelty of findings should be pointed out.

Author Response

Dear Reviewer,

herewith we submit the revised version of our manuscript by Filippone et al. We found your comments very helpful for improving the manuscript and included most of them in the present version. We outlined in detail how we addressed all your comments. We hope that this revised manuscript will meet your support and look forward to hearing from you.

Sincerely,

Giovanni Scopece

Department of Biology,

University Federico II of Naples,

Complesso Universitario MSA,

Via Cinthia, I-80126,

Naples, Italy

The manuscript agriculture-1782472 “Evaluation of Cadmium effects on six Solanum melongena L. 2 cultivars from the Mediterranean basin” try to identify pollution-safe cultivars (eggplant) with low translocation efficiency to aboveground organs to be used for food safety. The topic of this manuscript is meaningful and suitable for Agriculture Journal. However, this paper still need revision before accepted.

The critical points are reported below:

1. In the section of Introduction, Line 70-line 82, there are many papers research on screening for low-translocation efficiency (low accumulation) on agricultural plants. Please check the related studies on eggplant, if have, please added the introduction and references in this paragraph.

RESPONSE: Done, as suggested.

In the section of Materials and Methods, Line 116, why choose “15 days”, In my opinion, 15 days is just the growth period in eggplant. People eat the fruit of eggplant, harvest the eggplant when the fruit was growing will be more practical significance and evaluation value? Please explain it.

As correctly pointed out, our experimental design impedes us to detect Cd in the edible part of the plants, e.g. fruits, but allows to identify cultivars with low translocation efficiency to aboveground organs and to test the levels of expression of genes involved in Cd accumulation. Although this is not a direct estimation of Cd content in parts of the plant generally used for food consumption, leaves from young plants generally have a higher strength of fruits or seeds in terms of sink-source relationship. Accordingly, Shen et al. (2021) compared 30 eggplant cultivars and showed that in high Cd (H) and low Cd (L) accumulating cultivars Cd content in fruit was lower than Cd content in leaves. This suggests that Cd content in leaves from young plants might be used for a rapid screening of different cultivars. We added a brief statement in the revised discussions to acknowledge this point. In the conclusions we already mention the need of testing fruits in the more tolerant cultivars to further confirm the screening.

2. In the section of Materials and Methods, Line 131-142, the translocation factor and bio-concentration factor are most important index for evaluating heavy metal-tolerant plant. But in your equations, the bioaccumulation coefficient and translocation coefficients seem wrong. Please check the equations, and this article is for your reference: https://doi.org/10.1016/j.chemosphere.2021.130130

RESPONSE: We agree with the Reviewer’s comment and we thank her/him for spotting this issue. In the revised version we reformulated all the indices according to the reference suggested. We now also quote the reference in the text.

In the section of Materials and Methods, 2.8 statistical analysis. Please state n for Germination, Hydroponic culture, and qPCR.

RESPONSE: As suggested, we added in section 2.8 the number of individuals used for statistical analyses, for all the mentioned parameters.

3. In the section of Results, Line 248, “Cd 5 μM”? It should be “5 μM Cd”, please check across the whole manuscript.

RESPONSE: Done, as suggested

4. In the section of Results, Line 251, change mg Kg^-1 to mg kg^-1. And check across the whole manuscript.

RESPONSE: Done, as suggested

5. Figure 5, why some “※” was in the bar graph, some are out of bar graph? Please explain.

RESPONSE: We reported the meaning of stars in the Figure legend: “Statistical comparison was performed using a Student’s t-test, inside the bar plots are represented statistical differences with control samples, outside statistical differences between the two cultivars (* = P ≤ 0.05, ** = P ≤ 0.01; *** =P ≤ 0.001)”

8. In the section of Conclusions. The novelty of findings should be pointed out.

RESPONSE: In the revised version of the Conclusions we now point out the novelty of findings.

Round 2

Reviewer 2 Report

All my comments have been well responsed, and I have no additional comments.