Plant Growth and Nutrient Composition of Shrub and Arbor Willows Grown in Cu-Contaminated Flooded Soil

Round 1

Reviewer 1 Report

A very interesting work and one that requires special attention.

 I think this paper tried to draw attention to heavy metal pollution and to find cheap and environmentally friendly solutions. 

I have a few comments:

why was the abbreviation made like that? Salix jiangsuensis 'J172') and shrub willow (Salix integra 'Yizhibi')... wasn't it easier to code the whole paper?

 why was fe, mn determined if it was followed with?

 why was poplar leaf used as a control sample? why not willow?

 In Table 1, what do the letters a, b, c stand for, but "ns"? The table should be arranged so that certain data are not separated (e.g. Treatments× Spe......cies).

 What do NF and F stand for? no legend is given with these symbols

 In the discussion, comparisons should be made with other types of plants, e.g. : Adriana Mihaela. Chirilă Băbău,Valer Micle, Gianina Elena Damian, Ioana Monica Sur, Sustainable Ecological Restoration of Sterile Dumps Using Robinia pseudoacacia. Sustainability 2021, 13(24), 14021. https:// doi.org/10.3390/su1324140

 The conclusions are rather summary. They should probably be revised to highlight each parameter investigated.

Author Response

Reviewer #1

A very interesting work and one that requires special attention. I think this paper tried to draw attention to heavy metal pollution and to find cheap and environmentally friendly solutions.

Response: We thank the reviewer’s high evaluation on our manuscript.

 

I have a few comments:

1. why was the abbreviation made like that? Salix jiangsuensis 'J172' and shrub willow (Salix integra 'Yizhibi')... wasn't it easier to code the whole paper?

Response: Corrected. We use S. integra and S. jiangsuensis in the revised manuscript to avoid redundant.

 

2. why was fe, mn determined if it was followed with?

Response: Flooding forms soil anaerobic conditions and results in oxygen deficiency, which could provide the environment for Fe/Mn. oxide reduction/oxidation and sulfide formation. Flooding may further induce the formation of root plaque in some plants, which plays an important role in nutrient absorption or metal resistance.

 

3. why was poplar leaf used as a control sample? why not willow?

Response: Because poplar leaves are the best choice among the available plant standard sample we could selected (willow reference sample are not available or on sale).

 

4. In Table 1, what do the letters a, b, c stand for, but "ns"? The table should be arranged so that certain data are not separated (e.g. Treatments× Species).

Response: Different letters indicated significant difference among the 4 treatments (2 willow species under non-flooded and flooded conditions) at 0.05 level by Fisher's LSD test.

“ns” means not significant. We have included this in the revised manuscript.

 

5. What do NF and F stand for? no legend is given with these symbols

Response: NF and F represent the non-flooded and flooded conditions, respectively. We included the full name in the revised manuscript.

 

6. In the discussion, comparisons should be made with other types of plants, e.g. : Adriana Mihaela. Chirilă Băbău, Valer Micle, Gianina Elena Damian, Ioana Monica Sur, Sustainable Ecological Restoration of Sterile Dumps Using Robinia pseudoacacia. Sustainability 2021, 13(24), 14021. https:// doi.org/10.3390/su1324140

Response: Thanks. In the manuscript, we have compared willows with other plant species. For example: It was also suggested that the C contents were stable and not influenced by water supply/water depths in shrub Zygophyllum xanthoxylum and macrophyte species such as Potamogeton malaianus, Potamogeton maackianus, Myriophyllum spicatum, Ceratophyllum demersum and Hydrilla verticillate [29,59].

 

References

Niu, D.; Zhang, C.; Ma, P.; Fu, H.; Elser, J.J. Responses of Leaf C:N:P Stoichiometry to Water Supply in the Desert Shrub Zygophyllum Xanthoxylum. Plant Biol. 2019, 21, 82–88, doi:10.1111/plb.12897.

Li, W.; Cao, T.; Ni, L.; Zhang, X.; Zhu, G.; Xie, P. Effects of Water Depth on Carbon, Nitrogen and Phosphorus Stoichiometry of Five Submersed Macrophytes in an in Situ Experiment. Ecol. Eng. 2013, 61, 358–365, doi:10.1016/j.ecoleng.2013.09.028.

 

7. The conclusions are rather summary. They should probably be revised to highlight each parameter investigated.

Response: Agreed. We revised this section as the reviewer suggested.

Conclusions

Both willow species were able to grow well and showed relatively high tolerance to the combined stresses of Cu contamination and flooding. Flooding slightly decreased the total biomass (7.8–10.7%) of both willow species compared to non-flooded condition. Under flooded condition, S. integra showed greater potential of Cu accumulation capacity (1.39 mg per plant), even though its total biomass was significantly lower than S. jiangsuensis (arbor willow). Additionally, higher BCF values in roots (1.74) and root/shoot ratio (0.27) of S. integra were also observed in flooded treatment than that of S. jiangsuensis. Thus, S. integra was noted to be suitable for Cu phytostabilization with relative high Cu accumulation in roots regardless of flooding. Although characteristics of Cu accumulation differed between two willows, both species presented suitable tolerance mechanisms (regulating photosynthesis, nutrient uptake) to cope with flooding stress. Flooding markedly increased the C:P ratio in leaves of S. integra (69.7%) and S. jiang-suensis (44.4%), suggesting that flooding led to an elevated demand for assimilating more C to maintain the normal metabolisms in plants. Our study also helps further understanding the nutrient balance and stoichiometry of willows in Cu contaminated soil and their responses to soil flooding.

 

Author Response File: Author Response.docx

Reviewer 2 Report

 

Dear Authors!

The manuscript is devoted to the actual topic of soil phytoremediation, however, to improve its quality, I propose to make the following changes to it.

1. In Section 3.5, the numbering of lines has gone wrong, and it is missing in the Discussion section. This greatly complicates the quality review of the manuscript.

 2. I recommend pasting a more detailed description of how plants were grown under flood conditions in section 2.1.

3. Lines 126-129. The text does not refer to the leaf gas exchange measurement.

4. Line 138. Why was poplar leaf material used as a reference, and not willow, which was studied by the authors?

5. In the notes to the tables, it is necessary to give an explanation for the symbol “ns”, and in the captions to the figures, it is necessary to give an explanation for the symbol “NF” and “F”.

7. Table 1. Differences in wet weight of S. jiangsuensis ‘J172 leaves under and without flooding conditions are not significant.

8. Table S2 shows data on Zn, but the content of this element is not discussed in the manuscript.

9. I recommend making Fig.1 in color.

10. The numbering of Fig. 3 and 4 is confused.

11. Line 187. The text does not match the data in Fig.1.

11. The text of lines 191-194 does not match the data in Fig.1.

12. Line 223. According to Table 2 the increment of TF in S. jiangsuensis ‘J172’ was 272.73%, not 172.73%.

13. Section 3.5, lines 6-9. Phosphorus is missing from Table S4.

14. Is there a relationship between the content of C, N, P in the soil (Table S1) and the level of their accumulation in plants?

15. I recommend making references to figures and tables more often in the Discussion section.

16. Section 4.1. "After flooding for 90 d, all photosynthesis parameters were further decreased in both willow species, while Pn could maintain at a relatively high level." This text does not match the data in Fig.1.

17. The English language needs a lot of improvement.

Author Response

Reviewer 2

The manuscript is devoted to the actual topic of soil phytoremediation, however, to improve its quality, I propose to make the following changes to it.

1. In Section 3.5, the numbering of lines has gone wrong, and it is missing in the Discussion section. This greatly complicates the quality review of the manuscript.

Response: Corrected.

 

2. I recommend pasting a more detailed description of how plants were grown under flood conditions in section 2.1.

Response: Agreed. For flooded treatment, plants were grown in a depth of 10 cm water level from the soil surface, lasting for 3 months.

 

3. Lines 126-129. The text does not refer to the leaf gas exchange measurement.

Response: Corrected. We moved this part into other section (section 2.3).

 

4. Line 138. Why was poplar leaf material used as a reference, and not willow, which was studied by the authors?

Response: Because poplar leaves are the best choice among the available plant standard samples we could selected (willow reference sample are not available or on sale).

 

5. In the notes to the tables, it is necessary to give an explanation for the symbol “ns”, and in the captions to the figures, it is necessary to give an explanation for the symbol “NF” and “F”.

Response: Corrected. NF and F represent the non-flooded and flooded conditions, respectively. “ns” means not significant. We included the full name in the revised manuscript.

 

6. Table 1. Differences in wet weight of S. jiangsuensis ‘J172 leaves under and without flooding conditions are not significant.

Response: Here, we determined the dry weight (DW) not wet weight. For, S. jiangsuensis, no significance was observed in leaf biomass.

 

7. Table S2 shows data on Zn, but the content of this element is not discussed in the manuscript.

Response: Corrected. We deleted Zn in Table S2.

 

8. I recommend making Fig.1 in color.

Response: Thanks. We revised Fig.1 in color as the reviewer suggest.

 

 

9. The numbering of Fig. 3 and 4 is confused.

Response: Corrected.

 

10. Line 187. The text does not match the data in Fig.1.

Response: The result of interaction between flooding time and treatment was not available form Fig.1. We calculated this by two-way ANOVAs.

 

11. The text of lines 191-194 does not match the data in Fig.1.

Response: Disagreed. We double checked the data, and the results are accurate by calculating from the original data in Fig. 1.

 

12. Line 223. According to Table 2 the increment of TF in S. jiangsuensis ‘J172’ was 272.73%, not 172.73%.

Response: Disagreed. Here, we described the increment of TF, which calculated by (TF_flooded-TF_non-flooded)/TF_non-flooded 100%. If we described that TF under flooded condition is 272.73% of that under non-flooded condition, that is right.

 

13. Section 3.5, lines 6-9. Phosphorus is missing from Table S4.

Response: The data of P content was given in the main text in Table 4, not supporting information.

 

14. Is there a relationship between the content of C, N, P in the soil (Table S1) and the level of their accumulation in plants?

Response: We are sorry for not tested the changes of soil C, N, P. Here, we pay more attention to the nutrient changes in plant tissues.

 

15. I recommend making references to figures and tables more often in the Discussion section.

Response: Thanks. We revised this as the reviewer suggested.

 

16. Section 4.1. "After flooding for 90 d, all photosynthesis parameters were further decreased in both willow species, while Pn could maintain at a relatively high level." This text does not match the data in Fig.1.

Response: We are sorry for the inaccurate description. We deleted this sentence.

 

17. The English language needs a lot of improvement.

Response: Thanks. We further revised the language and improved both the quality and readability of the manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Page 4, line 159: it says: “ The Figureures were plotted…”. In my opinion, it should read: “The Figures were plotted …” The same error is found also in the legend of Figure 4; please change in this case "Figureure" to "Figure".

 

Please do not include the common names 'Yizhibi' and 'J172' every time Salix integra and Salix jiangsuensis are mentioned, respectively. Doing so the first time these taxa are mentioned in the main body of the article (e.g. in the introduction) is more than enough. Therefore, for naming both species throughout the text and the figures and tables, it is sufficient to cite them, for example, as S. integra and S. jiangsuensis, to avoid being too redundant. For example, the caption to Figure 1 reads …in leaves of S. integra (S. integra) ‘Yizhibi’ and S. jiangsuensis (S. jiangsuensis) exposed to Cu contaminated soil under …". In my opinion, it should read: “…in leaves of S. integra and S. jiangsuensis exposed to Cu contaminated soil under …...". Please extend this correction to the rest of the text.

 

The first time you include the Latin names of both species in the main text of the article (e.g. Introduction), you should include the full taxonomic designation of these names, including the authors of the Latin name.

 

Remember to always italicise the Latin names of all taxa mentioned in the article.

 

The caption to Table 1 should include the heading “Table 1” in bold.

 

Conclusions are too poor. They should be expanded to include more information derived from the research.

Author Response

Reviewer 3

1. Page 4, line 159: it says: “ The Figureures were plotted…”. In my opinion, it should read: “The Figures were plotted …” The same error is found also in the legend of Figure 4; please change in this case "Figureure" to "Figure".

Response: Corrected.

 

2. Please do not include the common names 'Yizhibi' and 'J172' every time Salix integra and Salix jiangsuensis are mentioned, respectively. Doing so the first time these taxa are mentioned in the main body of the article (e.g. in the introduction) is more than enough. Therefore, for naming both species throughout the text and the figures and tables, it is sufficient to cite them, for example, as S. integra and S. jiangsuensis, to avoid being too redundant. For example, the caption to Figure 1 reads …in leaves of S. integra (S. integra) ‘Yizhibi’ and S. jiangsuensis (S. jiangsuensis) exposed to Cu contaminated soil under …". In my opinion, it should read: “…in leaves of S. integra and S. jiangsuensis exposed to Cu contaminated soil under …...". Please extend this correction to the rest of the text.

Response: Thanks. We have corrected the related parts as the reviewer suggested.

 

3. The first time you include the Latin names of both species in the main text of the article (e.g. Introduction), you should include the full taxonomic designation of these names, including the authors of the Latin name.

Response: Corrected.

 

4. Remember to always italicise the Latin names of all taxa mentioned in the article.

Response: Corrected.

 

5. The caption to Table 1 should include the heading “Table 1” in bold.

Response: Corrected.

 

6. Conclusions are too poor. They should be expanded to include more information derived from the research.

Response: Thanks. We revised this section as the reviewer suggested.

Conclusions

Both willow species were able to grow well and showed relatively high tolerance to the combined stresses of Cu and flooding. Flooding slightly decreased the total biomass (7.8–10.7%) of both willow species compared to non-flooded condition. Under flooded condition, S. integra showed greater potential of Cu accumulation capacity (1.39 mg per plant), even though its total biomass was significantly lower than S. jiangsuensis (arbor willow). Additionally, higher BCF values in roots (1.74) and root/shoot ratio (0.27) of S. integra were also observed in flooded treatment than that of S. jiangsuensis. Thus, S. integra was noted to be suitable for Cu phytostabilization with relative high Cu accumulation in roots regardless of flooding. Although characteristics of Cu accumulation differed between two willows, both species presented suitable tolerance mechanisms (regulating photosynthesis, nutrient uptake) to cope with flooding stress. Flooding markedly increased the C:P ratio in leaves of S. integra (69.7%) and S. jiang-suensis (44.4%), suggesting that flooding led to an elevated demand for assimilating more C to maintain the normal metabolisms in plants. Our study also helps further understanding the nutrient balance and stoichiometry of willows in Cu contaminated soil and their responses to soil flooding.

 

Author Response File: Author Response.docx

Reviewer 4 Report

Today, the climate of the planet is changing greatly, causing both droughts and floods (respectively, new territories are flooded). If there are a lot of studies on the effects of drought on plants and their counteraction to it, the topic of plant stress caused by flooding remains less affected. And in conditions of heavy metal pollution (in particular copper ions) – practically unfilled, and, accordingly, requiring a solution. And there are prerequisites for solving this problem with the help of willow Salix.

            It is known that the absorption of copper by plants significantly depends on the pH of the medium, it increases with a decrease in pH, on the contrary, with an increase in the value, the total solubility and activity of copper ions decreases. The presence of humus, N, P affects the availability of copper when absorbed by the root system. Accordingly, the mobility of copper and its availability to plants in your conditions pH (6,9) is not optimal. Or did the pH change somehow during the course of the experiment? How can humus, N, P of the soil affect the availability of copper ions to plants?

            It is also of interest to study the interaction of copper ions with other heavy metals, which reduce (?) its toxic effect. Why were the heavy metals presented evaluated, and not, for example, cadmium, lead? What is their initial content in the soil? (no data provided). Correlation with the original content? Did they exert additional stress?

            The topic of the article is copper. Why was the accumulation of other elements (Mg, S, Ca, Fe, Mn, etc.) considered? What is the significance of the presence of above normal values or deficiency of macro and microelements on the accumulation of copper in the studied plants?

-Are there any characteristics of tap water used for irrigation?

-50% of references older than 10 years.

- Why are studies limited to only two types of willow??

 

44: It is required to explain the spread of values of accumulated copper.

98: Isn't the volume of the container for growing willow too small?

101-112: How many plants were taken for each variant? (How many repetitions?)

 

Table 1. Absolute values of flooded and non-flooded plants differ slightly, and taking into account errors, the values overlap.

 

3.2. Leaf gas exchange

Measuring the level of moisture transpiration and gas exchange in flooded plants in comparison with non-flooded ones, taking into account the difference in root systems between them, does not seem quite correct.

 

P.S. Is this research already presented in full-text form on the web?

·         DOI:10.21203/rs.3.rs-689584/v1

·         Corpus ID: 238645162

Plant growth and nutrient composition in shrub and arbor willows grown in Cu contaminated soil as affected by flooding

·         Yini Cao, Chuanxin Ma, Jie Chen, Jiang Xiao, Jiuxi Shi, Guangcai Chen less

·         Published 11 August 2021

·         Environmental Science

Author Response

Reviewer 4

Today, the climate of the planet is changing greatly, causing both droughts and floods (respectively, new territories are flooded). If there are a lot of studies on the effects of drought on plants and their counteraction to it, the topic of plant stress caused by flooding remains less affected. And in conditions of heavy metal pollution (in particular copper ions) – practically unfilled, and, accordingly, requiring a solution. And there are prerequisites for solving this problem with the help of willow Salix. 

Response: Thanks. Although numerous studies have investigated the metal or flooding stress on phytoremediation efficiency, most of them focused on only one stress factor, and the joint effect (co-exposure of heavy metal and flooding) on woody plants has not been sufficiently studied.

 

It is known that the absorption of copper by plants significantly depends on the pH of the medium, it increases with a decrease in pH, on the contrary, with an increase in the value, the total solubility and activity of copper ions decreases. The presence of humus, N, P affects the availability of copper when absorbed by the root system. Accordingly, the mobility of copper and its availability to plants in your condition pH (6.9) is not optimal. Or did the pH change somehow during the course of the experiment? How can humus, N, P of the soil affect the availability of copper ions to plants?

Response: This is good question. The changes of soil pH were given in the following Table. Significant decrease of soil pH was observed between flooded and non-flooded conditions.

Willow species	Non-flooded	Flooded
S. integra	6.93 0.11b	6.74 0.05c
S. jiangsuensis	7.1 0.3a	6.70 0.07c

 

We are sorry for not tested the changes of soil C, N, P. Here, we pay more attention to the nutrient changes in plant tissues. In the future study, we will do more work on the relationship between soil nutrients and metal availability.

 

It is also of interest to study the interaction of copper ions with other heavy metals, which reduce its toxic effect. Why were the heavy metals presented evaluated, and not, for example, cadmium, lead? What is their initial content in the soil? (no data provided). Correlation with the original content? Did they exert additional stress?

Response: Here, the collected soil was heavily contaminated by Cu but not other metals (e.g. Pb or Cd). The other metal contents were extremely lower than the risk screening values for soil contamination, which were not toxicity for plant growth. Thus, these metals were not considered and discussed in the manuscript.

 

The topic of the article is copper. Why was the accumulation of other elements (Mg, S, Ca, Fe, Mn, etc.) considered? What is the significance of the presence of above normal values or deficiency of macro and microelements on the accumulation of copper in the studied plants?

Response: Flooding forms soil anaerobic conditions and results in oxygen deficiency, which could provide the environment for Fe/Mn. oxide reduction/oxidation and sulfide formation. Flooding may further induce the formation of root plaque in some plants, which plays an important role in nutrient absorption or metal resistance.

Under flooded conditions, plants exhibit different strategies for nutrient uptake and utilization, and the alteration could lead to the differences in elemental stoichiometry. Thus, it is essential to investigate the macro nutrients in plants to explain how plant in response to flooding and metal stresses.

 

Are there any characteristics of tap water used for irrigation?

Response: We did not test the parameters of tap water. Tap water is fit for water quality standards, and pollutants can be neglected. Thus, it will not affect the normal growth of plants

 

50% of references older than 10 years.

Response: Agreed. We have renewed some of the references.

 

-Why are studies limited to only two types of willow?

Response: In our previous studies, we investigated the Cu accumulation capacity of shrub willow (Salix integra ‘Yizhibi’) and arbor willow (Salix jiangsuensis ‘J172’) at different Cu levels (including the control soils without Cu contamination) under non-flooded and flooded conditions (Cao et al., 2019; Chen et al., 2013), respectively. The results illustrated that these two willows are good candidates for Cu-contaminated soil as affected by soil flooding. However, our earlier studies conducted with artificially added Cu soils, which might not reflect the real circumstance of contaminated site. Thus, we collected the Cu-contaminated soil from the local field to compare the flooding influence on these two willows. In the present manuscript, we mainly focused on how soil flooding affected the Cu phytoremediation capacity and nutrient composition, and then further screened the phytoremediation candidate of willows and discussed their underlying mechanisms by which Cu stress altered the nutrient balance in willow. We believe that the current study is a step further based on our previous studies and can help to understand the phytoremediation of both willow species under flooding conditions.

References

Cao YN, Ma CX, Zhang JF, Wang SF, White JC, Chen GC, Xing BS (2019) Accumulation and spatial distribution of copper and nutrients in willow as affected by soil flooding: A synchrotron-based X-ray fluorescence study. Environ Pollut 246: 980–989.

Chen GC, Liu ZK, Zhang JF, Owens G (2013) Phytoaccumulation of copper in willow seedlings under different hydrological regimes. Ecol Eng 44: 285–289.

 

44: It is required to explain the spread of values of accumulated copper.

Response: Here, the Cu contents was not by Cu spike, it is the real contents collected from the local area.

 

98: Isn't the volume of the container for growing willow too small?

Response: Thanks. Maybe we could change a larger container for planting next time.

 

101-112: How many plants were taken for each variant? (How many repetitions?)

Response: Four replicates for each treatment.

 

Table 1. Absolute values of flooded and non-flooded plants differ slightly, and taking into account errors, the values overlap.

Response: Disagreed. The average values are completely different. There exhibit significant differences between non-flooded and flooded in total biomass.

 

3.2. Leaf gas exchange 

Measuring the level of moisture transpiration and gas exchange in flooded plants in comparison with non-flooded ones, taking into account the difference in root systems between them, does not seem quite correct.

Response: Thanks. We deleted this sentence.

 

P.S. Is this research already presented in full-text form on the web?  DOI:10.21203/rs.3.rs-689584/v1

Corpus ID: 238645162  Plant growth and nutrient composition in shrub and arbor willows grown in Cu contaminated soil as affected by flooding  Yini Cao, Chuanxin Ma, Jie Chen, Jiang Xiao, Jiuxi Shi, Guangcai Chen  Published11 August 2021  Environmental Science

Response: The pre-print on the Research Square can be found as described. It should be stated that the pre-print was guided and uploaded during our previous submission. It is accepted by public that the pre-print was encouraged to spread the findings. Finally, we declare that we have no multiple submissions with the same manuscript to different journals.

Author Response File: Author Response.docx

Round 2

Reviewer 4 Report

Thanks for the answers and corrections.