Transcriptome Analysis Reveals Key Genes and Pathways Associated with Cadmium Stress Tolerance in Solanum aculeatissimum C. B. Clarke

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study on 'Transcriptome analysis reveals key genes and pathways associ-ated with cadmium-stress tolerance in Solanum aculeatissimum' has been conducted very well with proper experimentation and data. Overall it is written well but can be improved with some language corrections. I have few comments to be addressed by authors.

1. Why authors selected the plant, S. aculeatissimum , a weedy plant for their study. What are the unique characters of this plant can be provided to justify.

2.Section 2.3 showed that authors used 'Rectangular plastic pots', but Fig 1 showed they are round pots.

3.Table 2, 3 showed changes in plant herght and stem length. what is the reason of taking stem length when total plant height is taken already. 

4.Surprisingly Cd (Cd1-Cd 5) has shown an increase in both the parameters. Normally, p-lants will be affected by Cd level. Can authors explain this growth increase

5.Table 4.  has several quantitative data in the form of number of flowers, fruits, fruit diameter. Table should be modified to indicate such observations.

6.Authors can explain why they use 'uptake enrichment-related gene screening', when it is actually 'upregulation'. 

Comments on the Quality of English Language

English language can be improved

Author Response

Thank you for your response, 6 questions have been revised in the manuscript. Here is a description of the revisions.

The study on 'Transcriptome analysis reveals key genes and pathways associ-ated with cadmium-stress tolerance in Solanum aculeatissimum ‘has been conducted very well with proper experimentation and data. Overall it is written well but can be improved with some language corrections. I have few comments to be addressed by authors.

 

1. Why authors selected the plant, S. aculeatissimum , a weedy plant for their study. What are the unique characters of this plant can be provided to justify.

 

Solanum Aculeatissimum is a plant of the Solanaceae family that has been found to grow normally in lead-zinc mining districts.Deng Xiaopeng found that Solanum nigrum showed a strong accumulation capacity of Cd, Pb, Mn, the maximum accumulation of leaves reached 145.9, 391.2 and 5239 mg/kg; Lycopersicon esculentum has a certain degree of tolerance to Cd, Pb, able to prevent or exclude the absorption of it; Solanum indicum has a strong transfer capacity of Cd, the highest Cd content of the leaves reached 46.62mg/kg, the ratio of Cd content in leaves/roots reached 6.18, while able to enrich a large amount of Pb in the roots, up to 207mg/kg; pepper above ground can accumulate a certain amount of Cd, up to 42.71mg/kg, for other elements, the maximum Cd content of 207mg/kg; Capsicum annuum above ground can accumulate a certain amount of Cd, up to 42.71mg/kg, for the other elements. The maximum Cd content of leaf was 46.62mg/kg, and the ratio of leaf/root Cd content was as high as 6.18, meanwhile, it could enrich a large amount of Pb in the root, up to 207.1mg/kg; C. annuum could accumulate a certain amount of Cd above ground, up to 42.71mg/kg; S. nigrum and S. indicum could be used for phytoremediation of the main soil infected with heavy metals.

We found that plants of the Solanaceae family can be well adapted to the environment polluted by lead and cadmium , and we chose S. Aculeatissimum as our test material.

 

2.Section 2.3 showed that authors used 'Rectangular plastic pots', but Fig 1 showed they are round pots.

 

Fig. 1 shows the use of round plastic is used for planting plants of three months old saplings. In the experiment of cadmium stress contamination of plants, we moved the three months old saplings to rectangular plastic pots

 

3.Table 2, 3 showed changes in plant herght and stem length. what is the reason of taking stem length when total plant height is taken already.

 

The authors' personal understanding is that the phenotype of a plant consists of plant height and stem thickness, with plant height representing vertical data and stem thickness representing horizontal data.

 

4. Surprisingly Cd (Cd1-Cd 5) has shown an increase in both the parameters. Normally, plants will be affected by Cd level. Can authors explain this growth increase

 

In a short period of time (15 days), cadmium stress at a moderate concentration (Cd1) was able to promote the height of S. Aculeatissimum which is consistent with the stress characteristic of "low promotion and high inhibition".

At the higher concentration (Cd5), the height of the plants reached a high level, and at the highest concentration (Cd6), the height of the plants declined, which I guessed to be the threshold value of cd stress in the concentration range of Cd5 and Cd6.

 

5. Table 4.  has several quantitative data in the form of number of flowers, fruits, fruit diameter. Table should be modified to indicate such observations.

 

The author does not quite understand how these quantitative data should be better described and would appreciate better guidance from the reviewers.

 

6. Authors can explain why they use 'uptake enrichment-related gene screening', when it is actually 'upregulation'.

 

Under the same stress conditions, the screened genes showed "up-regulation", indicating an increase in gene expression of the genes concerned, and the trend of Cd content in the determined plants was consistent in different parts (roots and stems).

Therefore, it is suggested that the expression of the selected genes increased with the increase of Cd content in the plants. The authors believe that the up-regulation and down-regulation of the screened genes are relative to the fact that the expression of the genes in different parts (roots and stems) did not increase or decrease with the increase of Cd concentration in plants.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Brief summary

This manuscript aimed to elucidate key genes and pathways associated with cadmium-stress tolerance in Solanum aculeatissimum. The authors concluded that Cd-treated S. aculeatissimum had no inhibitory growth (plant height, stem height, and number of leaves). The authors also recorded the Cd concentrations within the various plant organs at 2 time points, indicating high Cd enrichment and translocation rates, thus providing evidence of S. aculeatissimum’s ability to be used in remediating Cd-contaminated soils. The authors assessed the damaging effect of Cd on S. aculeatissimum by determining MDA content and S. aculeatissimum’s response to damage by investigating three antioxidants (SOD, CAT, and POD). The authors lastly identified genes that were differentially expressed under Cd stress, however, could not elucidate their roles in Cd stress due to limited literature.

 

Comments and Suggestions:

1.      In line 13, cadmium is mentioned for the first time, therefore, authors should write out the full name first, and thereafter the authors can use the acronym

2.      What do the authors mean when they indicate that S. aculeatissimum was ‘well tolerant’ in line 17?

3.      What are the authors attempting to say when using the word ‘enriched’ in line 17? I think this is incorrect use of the word.

4.      I suggest the authors use an alternative word to ‘size’ in line 19. Use ‘amount’ or ‘concentration’.

5.      In Lines 20-23 the authors mention various plant organs and the Cd content therein and at what Cd concentration they peaked, however, 6 organs were mentioned but only 5 Cd concentrations were respectively mentioned. The authors should correct this result.

6.      Authors should use an appropriate word (relevance) in line 30.

7.      In lines 21-23, the authors mention mature fruit Cd content result, however, reviewing the result section no mention or figure illustrating the mature fruit Cd concentration. Authors should thus review their result section or remove the mention of mature fruit Cd content in line 23.

8.      Incorrect word use (necessary) in line 37.

9.      Remove the comma in line 38 and replace it with a full stop. The two sentences are two different statements and therefore should be two separate sentences.

10.   What do the authors want to convey when they say ‘Cd is a representative heavy metal…’ in line 38.

11.   Correct the word ‘development’ to ‘developmental’ in line 39.

12.   Throughout the manuscript correct the in-text reference brackets.

13.   Lines 41-49, could be condensed into a shorter explanation, as there are many repetitive statements being made within these lines.

14.   Lines 52-55 is too long.

15.   Lines 55-59 is too long.

16.   ‘Reverses’ in line 60 might be incorrectly used.

17.   Incorrect use of the word ‘dwarf ’ in line 67.

18.   In line 67 the authors indicated that Cd stress results in ‘greenish’ leaves. Is this the case, many studies have provided evidence that Cd stress results in decreased chlorophyll content (yellowing of the leaves) and photosynthetic activity.

19.   The reference Ling et al., 2017 indicated a decrease in chlorophyll contents in the leaves of corn with an increase in Cd supply. Line 67. However, the authors within this manuscript indicate high Cd content results in greenish leaves which contradicts the Ling et al., 2017 study.

20.   What is meant by ‘significant concentration effect’ in line 70?

21.   Lines 78-79 authors should decide whether they will write either the common name or scientific names of plants here and throughout the manuscript. In lines 78-79, the authors use both.

22.   See the above comment for ‘Southeast sedum’ and ‘Populus ’ in lines 82 and 83.

23.   Authors should separate listed items with a comma throughout the manuscript. An example of such an issue is presented in line 85 and lines 91-93.

24.   What are the authors trying to convey when saying ‘Tolerance absorption’ in line 95?

25.   Is it necessary for ‘for heavy metals’ in line 95, as the authors already stipulated for Cd (line 95).

26.   The authors should replace ‘heavy metals’ with ‘Cd’ in line 98, as their study was focused on only investigating Cd and not any other heavy metal.

27.   Authors should be careful when referring to the sterilization of seeds. In lines 108 and 110 authors should say ‘surface sterilized’ and not ‘sterilized’. Just saying sterilized gives the impression that the seeds were sterilized meaning killed.

28.   Were two sterilization methods used in the investigation or was the gibberellin and water bath soaking an additional step to the previously mentioned surface sterilization (Lines 108-111)?

29.   Authors should reference where they obtained the surface sterilization method.

30.   Authors should mention how the physiochemical properties of the test soils were achieved (ex. What approach was used? and which equipment was used?) (line 116).

31.   Authors should correctly align all contents in Table 1, especially for the pH value.

32.   Authors should correct ‘7 treatment groups’ to ‘6 treatment groups’ as only 6 Cd treatment groups are indicated alongside the control group (CK) (Line 127-129).

33.   Once again authors should revise the amount of Cd treatments set up. Because line 130 mentions ’42 pot were treated with Cd heavy metal’. If 6 pots of each treatment was set up for each Cd treatment, that would indicate that there was a range of 7 different Cd treatments set up besides the control (CK) and that is not the case. 6 Cd treatments are mentioned in the investigation.

34.   Line 132-133, revise the sentence to make it more comprehensive.

35.   Line 133, Correct the beginning of the sentence.

36.   Authors should provide the actual growth parameters (ex. Temperature) in which they grew their S. aculeatissimum plants (line 133), for reproducibility purposes.

37.   Is Figure 1 stretched?

38.   Similar comment as prior, authors should be specific. The investigation only focused on Cd stress, thus authors should replace heavy metals in line 138 with Cd.

39.   Similar comment as prior. In line 138, authors should revise their treatment groups and how they mention them.

40.   Replace ‘days’ with ‘day’ in line 145.

41.   Suggestion: remove ‘respectively’ in line 145.

42.   Correct days at the beginning of line 153.

43.   In line 158 authors are confusing as first they state ‘taken from the top’, then thereafter they state ‘counting down from the first fully expanded leaf’. Authors should clearly state which leaves were used.

44.   Line 160, see previous comment 45.

45.   Is ‘and extracted them’ necessary in line 163.

46.   Authors should clarify what is meant by ’10 % TCA ice bath’ in line 164.

47.   Should the Item No. for CAT and POD to be switched (lines 166-168)?

48.   Please clarify what ‘extract’ (line 168) was used. To what are the authors referring to when mentioning ‘extract’

49.   The methodology for antioxidant determination (SOD, CAT, and POD) needs to be revised to ensure that it can be reproducible (lines 166-171).

50.   Why did authors conduct Cd concentration determination after the 60^th and 163^rd days of Cd treatment, and antioxidant and MDA after the 90^th and 163^rd days of Cd treatment? To compare and make an accurate conclusion the same age of plant material should be used. Authors should also provide the exact age (days) of the material used.

51.   Authors should stipulate how the different plant organs were dried (line 175).

52.   Rephrase lines 177-178.

53.   What is an acid-depleting apparatus? And what is the purpose of it in this assay? (lines 179-180).

54.   Authors should rephrase lines 181 to 183.

55.   Authors should mention what solution was used as the blank control in line 184, for reproducibility purposes.

56.   Once again why was after 60 days used for cDNA construction? What exact day plant material was used? Mentioning after 60 days could mean either 90 days or 163 days.

57.   Did the authors mean ‘from the whole pot’ in line 193?

58.   Were the plant organs of the same treatment all merged into one tube (lines 195-197). Authors should be clear in their entire methodology section.

59.   The subheading 2.8 does not match the description of the methodology there under lines 192-197.

60.   Authors should mention whether they followed the manufacturer’s instructions when using the Total Plant RNA Extraction Kit (lines 199-200).

61.   In line 212, FPKM is the first mention, thus authors should write out the full acronym at first mention.

62.   Lines 222-224 are fragmented.

63.   Authors should clarify whether 5 primers were synthesized for each, or how was the distribution of synthesized primers (lines 231-232).

64.   Lines 234-239 explain the RNA extraction method. Should this method not have been explained in an earlier section (section 2.9)?

65.   Italicize ‘S.aculeatissimum’ in line 247

66.   Authors should be specific to their investigation. Replace ‘heavy metal’ in line 249 with ‘Cd’.

67.   Throughout the manuscript authors should pay attention to the way they outline their treatment groups and the phrasing thereof.

68.   Why did the authors not consider investigating the phenotype of the rooting system? The authors spoke about the rooting system being the main entry point serving its importance in Cd uptake as well as authors investigated the rooting material in all other assays within the investigation (line 250).

69.   Lines 250-251, the authors perfectly elucidated the number of treatment groups (6 different Cd concentrations). Authors should thus correct all the other mentions of 7 Cd-treatment groups.

70.   Correct the diagonal line in both Table 2 and Table 3.

71.   The alignment of the entire Cd6 row in Table 2 and Table 3 needs to be correct.

72.   Clear descriptions in the first cell in both Table 2 and Table 3 should be provided.

73.   Why is the plant growth only measured up to 60 days? Some of the other assays are conducted on much more mature plants. Where are the measurements for those days (days 90 to 163)?

74.   Information about Notes of tables above line 281 is not for the table but for the Figure. Thus correct ‘The results in the Table’ to ‘The results in the Figure’.

75.   Stem thickness in line 255 is not shown in the results.

76.   This is not true for Cd1, Cd4, Cd5, and Cd6 (line 258).

77.   Authors should be consistent with what they are referring to when mentioning the result regarding the stem morphology. The Table indicates Stem length, whereas in the text the authors keep inter-changing stem length and stem height (line 259).

78.   Check alignment in each table in the entire manuscript.

79.   Authors should rephrase lines 289-291. Change ‘The variation of S. aculeatissimum content’ because authors are testing Cd content in S. aculeatissimum.

80.   All sub-images in Figure 3 have a labeled scale except Figure 3B. Please correct this.

81.   Authors should increase the font of axis labels in Figure 3, to make it more legible.

82.   The labeling for the control group should be kept consistent throughout the manuscript. The authors depicted the control group as CK however, in Figure 3, the control group is written as Cd0. To avoid confusion authors should change this Cd0 to CK.

83.   In Figure 3C the last bar for Cd6 is cut off.

84.   In Figure 3C, the authors represent roots, stems, leaves, unripe fruit, and ripe fruits, however, in the abstract when referring to this section of results, the author also mentioned mature fruit which is not presented here. Authors should consider either adding this result in the result section or removing the mention of the mature fruit.

85.   Throughout the manuscript the authors refer to the enrichment coefficient, however in Table 5 it is referred to as the Bioconcentration factor. Authors should please be consistent in the terminology used, whether in text or Figures and Tables.

86.   When referring to the transport coefficient in Section 3.2.2 are the authors referring to the translocation of Cd or what are the authors referring to?

87.   As mentioned previously, authors should review the Figures and the font sizes of the axis to make them more legible.

88.   Authors should also keep the Figures' labelling layout consistent throughout the manuscript. Figure 3 The Figure did not have a heading and the y-axis had more information whereas in Figure 4 the Figures have headings and the y-axis labelling only consists of SI units.

89.   Figure 4 legend should indicate on what day these measurements were taken. The figure legend should also indicate what plant organ these results were extrapolated from.

90.   The authors mention in section 3.4 that the average GC content was 48.38 %, however, Table 6 depicts a different value (46.90).

91.   The author also keeps mentioning ‘three samples’ however in the Tables only two samples are presented) (Ck and Cd6).

92.   Authors should correct Table 7 so that it is a stand-alone Table from Table 6.

93.   Once again Authors should revise the font for Figures.

94.   Figure 5 legend only mentions the Volcano plot, however, the Figure is of both a Volcano plot and a Scatter plot.

95.   The authors mention the 5 highest DEG groups however, what the authors listed does not correlate to the Figure. The 5 highest DEG groups appear to be Global and overview maps, Signal transduction, immune system, carbohydrate metabolism, and amino acid metabolism (Lines 364-365).

96.   Where are the results that correlate with section 3.8? where is this information extrapolated from?

97.   What do the authors mean when saying ‘results of S. aculeatissimum content’ in lines 405-406? What content is being referred to? The authors should be specific.

98.   Authors mention ‘SaACs4’ in line 408, however, this gene was not represented in the figure.

99.   Is position the correct term to use? Should authors not refer to the x-axis as plant organs?

100.                    Provide a reference for the first statement in the discussion (lines 411-413).

101.                    What do the authors mean when they say’ change in height and stem height of S. aculeatissimum was not the same’ in line 417?

102.                    The authors once again refer to stem thickness in line 423. Are the authors correlating stem height, stem length, and stem thickness? Because Stem thickness is not presented in the result section.

103.                    In lines 428-430, the authors mention that increased Cd promoted leaf number, which was the case in their investigation, compared to their control. However, the authors go further in saying that with prolonged stress time, an inhibitory effect was seen on the leaves, which was not the case. The majority of the Cd treatments from day 15- day 90 illustrated more leaves than the control group (Figure 2).

104.                    Rephrase lines 445-447, as the first half of the sentence makes it sound like MDA decreases with an increase in Cd concentration, which is not the case.

105.                    In lines 452-453, the authors mention the MDA content of the roots and leaves, however, the only result focused on MDA (Figure 4 A) does not stipulate which plant organ it is conducted on. Please correct this.

106.                    The authors mention the studies in lines 447-452 however do not compare this study with their current results.

107.                    Lines 455-457, are the authors referring to their current investigation? Or are the authors referring to another study that was conducted?

108.                    The conclusion in lines 465-468 cannot be made without the supporting evidence. The authors did not conduct hydrogen peroxide determination so authors cannot say that hydrogen peroxide was cleared by CAT. The same can be said with the statement in line 465, no ROS determination assays were conducted thus authors cannot conclude that there was excessive ROS produced they can only postulate this.

109.                    Once again Figure 4 did not stipulate the organ that was used for this antioxidant determination, however in lines 473-474 the authors mentioned leaves.

110.                    The authors cannot make conclusive remarks about antioxidants and their activity over prolonged periods of Cd stress as they did not include the MDA measurements at 163 days. In this investigation, authors could only extrapolate conclusive remarks from day 90.

111.                    In line 489 authors did not previously mention these acronyms. Thus, it is suggested that the authors write out the full words and then include the acronyms.

112.                    Provide references for lines 491-493.

113.                    In lines 518-519, there is repetition at the beginning and end of this sentence.

114.                    The authors mentioned the KEGG results, and the genes however did not give a brief explanation as to the importance of these genes about stress. Were they involved in Cd stress response or Cd stress tolerance?

115.                    The authors mention ‘The main antioxidant systems involved include antioxidant enzymes, glutathione 521 transferase, oleuropein lactone biosynthesis, and hydrolase activity’ in lines 521-522. Under what conditions are these antioxidants the main antioxidant systems? Furthermore, what is the purpose of mentioning this if none of these antioxidants were investigated in the current study? Why did the authors not investigate these antioxidants if they are referred to as the main antioxidant systems?

116.                    Paragraphs 521-530, refer to genes related to antioxidant systems, however, nowhere in the manuscript do the authors mention or present results elucidating or identifying any genes involved in the antioxidant systems. The only results provided for antioxidants refer to the activity of antioxidant enzymes.

117.                    In lines 547-554, authors should use the literature they mentioned (lines 547-552) to conclude or explain their results in lines 552-555.

118.                    Correct the font of ‘Nightshade’ in line 575.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The use of the English language needs to be improved, this is evident in the sentence structures and multiple occasions of incorrect word usage. Hence the manuscript requires major and thorough revision writing, language use, and consistent checks and reformatting of references. Unfortunately, I am unable to give extensive point-by-point review comments on language. 

Author Response

Thanks to the reviewers for asking me 118 questions, I answered each one and made changes directly in the paper!I don't quite understand three of them(No.75,No.76,No117.), and would appreciate further explanation.Thank you very much！（I need to fill in three additional tables）

Comments and Suggestions:

 

1. In line 13, cadmium is mentioned for the first time, therefore, authors should write out the full name first, and thereafter the authors can use the acronym

    Modified.

 

2. What do the authors mean when they indicate that S. aculeatissimum was ‘well tolerant’ in line 17?

The term "well tolerant" is used by the author to mean that under high levels of Cd stress, S. aculeatissimum is well adapted and grows normally with no mortality.

3. What are the authors attempting to say when using the word ‘enriched’ in line 17? I think this is incorrect use of the word.

Modified.

4. I suggest the authors use an alternative word to ‘size’ in line 19. Use ‘amount’ or ‘concentration’.

Modified.

5. In Lines 20-23 the authors mention various plant organs and the Cd content therein and at what Cd concentration they peaked, however, 6 organs were mentioned but only 5 Cd concentrations were respectively mentioned. The authors should correct this result.

Modified.

6. Authors should use an appropriate word (relevance) in line 30.

Modified.

7. In lines 21-23, the authors mention mature fruit Cd content result, however, reviewing the result section no mention or figure illustrating the mature fruit Cd concentration. Authors should thus review their result section or remove the mention of mature fruit Cd content in line 23.

Cadmium content of mature fruits is shown in manuscript P10 figure 3.

8. Incorrect word use (necessary) in line 37.

Modified.

9. Remove the comma in line 38 and replace it with a full stop. The two sentences are two different statements and therefore should be two separate sentences.

Modified.

10. What do the authors want to convey when they say ‘Cd is a representative heavy metal…’ in line 38.

The authors expressed the idea that cadmium contamination is representative of heavy metal contamination in soil.

11. Correct the word ‘development’ to ‘developmental’ in line 39.

Modified.

12. Throughout the manuscript correct the in-text reference brackets.

Modified.

13. Lines 41-49, could be condensed into a shorter explanation, as there are many repetitive statements being made within these lines.

Modified.

14. Lines 52-55 is too long.

Modified.

15. Lines 55-59 is too long.

Modified.

16. ‘Reverses’ in line 60 might be incorrectly used.

Modified.

17. Incorrect use of the word ‘dwarf ’ in line 67.

Modified.

18. In line 67 the authors indicated that Cd stress results in ‘greenish’ leaves. Is this the case, many studies have provided evidence that Cd stress results in decreased chlorophyll content (yellowing of the leaves) and photosynthetic activity.

Modified.

19. The reference Ling et al., 2017 indicated a decrease in chlorophyll contents in the leaves of corn with an increase in Cd supply. Line 67. However, the authors within this manuscript indicate high Cd content results in greenish leaves which contradicts the Ling et al., 2017 study.

Modified..The translation ‘greenish’is wrong. It's trying to say that the leaves are not green and turn yellow.

20. What is meant by ‘significant concentration effect’ in line 70?

There was a significant difference between different concentrations of cd stress on chili pepper yield.

21. Lines 78-79 authors should decide whether they will write either the common name or scientific names of plants here and throughout the manuscript. In lines 78-79, the authors use both.

Modified.

22. See the above comment for ‘Southeast sedum’ and ‘Populus ’ in lines 82 and 83.

Modified.

23. Authors should separate listed items with a comma throughout the manuscript. An example of such an issue is presented in line 85 and lines 91-93.

Modified.

24. What are the authors trying to convey when saying ‘Tolerance absorption’ in line 95?

Written by the author in error, trying to describe tolerance, absorption and enrichment.Modified.

25. Is it necessary for ‘for heavy metals’ in line 95, as the authors already stipulated for Cd (line 95).

Modified.

26. The authors should replace ‘heavy metals’ with ‘Cd’ in line 98, as their study was focused on only investigating Cd and not any other heavy metal.

Modified.

27. Authors should be careful when referring to the sterilization of seeds. In lines 108 and 110 authors should say ‘surface sterilized’ and not ‘sterilized’. Just saying sterilized gives the impression that the seeds were sterilized meaning killed.

Modified.

28. Were two sterilization methods used in the investigation or was the gibberellin and water bath soaking an additional step to the previously mentioned surface sterilization (Lines 108-111)?

Erythromycin and water bath soaking serve to promote seed germination and are additional steps to the surface sterilization mentioned previously.

29. Authors should reference where they obtained the surface sterilization method.

Modified.

30. Authors should mention how the physiochemical properties of the test soils were achieved (ex. What approach was used? and which equipment was used?) (line 116).

Modified.

31. Authors should correctly align all contents in Table 1, especially for the pH value.

Modified.

32. Authors should correct ‘7 treatment groups’ to ‘6 treatment groups’ as only 6 Cd treatment groups are indicated alongside the control group (CK) (Line 127-129).

Modified.

33. Once again authors should revise the amount of Cd treatments set up. Because line 130 mentions ’42 pot were treated with Cd heavy metal’. If 6 pots of each treatment was set up for each Cd treatment, that would indicate that there was a range of 7 different Cd treatments set up besides the control (CK) and that is not the case. 6 Cd treatments are mentioned in the investigation.

Modified.

34. Line 132-133, revise the sentence to make it more comprehensive.

Modified.

35. Line 133, Correct the beginning of the sentence.

Modified.

36. Authors should provide the actual growth parameters (ex. Temperature) in which they grew their S. aculeatissimum plants (line 133), for reproducibility purposes.

Modified. In line 162-167.

37. Is Figure 1 stretched?

Modified.Have reset the image size.

38. Similar comment as prior, authors should be specific. The investigation only focused on Cd stress, thus authors should replace heavy metals in line 138 with Cd.

Modified.

39. Similar comment as prior. In line 138, authors should revise their treatment groups and how they mention them.

Modified.

40. Replace ‘days’ with ‘day’ in line 145.

Modified.

41. Suggestion: remove ‘respectively’ in line 145.

Modified.

42. Correct days at the beginning of line 153.

Modified.

43. In line 158 authors are confusing as first they state ‘taken from the top’, then thereafter they state ‘counting down from the first fully expanded leaf’. Authors should clearly state which leaves were used.

Modified.

44. Line 160, see previous comment 45.

Modified.

45. Is ‘and extracted them’ necessary in line 163.

Modified.

46. Authors should clarify what is meant by ’10 % TCA ice bath’ in line 164.

Modified.

47. Should the Item No. for CAT and POD to be switched (lines 166-168)?

Modified.

48. Please clarify what ‘extract’ (line 168) was used. To what are the authors referring to when mentioning ‘extract’

Extract is the “kit xtract”.The ingredients of the "kit extract" are not specified in the kit manual.

49. The methodology for antioxidant determination (SOD, CAT, and POD) needs to be revised to ensure that it can be reproducible (lines 166-171).

Modified.Iine 189-198.

50. Why did authors conduct Cd concentration determination after the 60th and 163rd days of Cd treatment, and antioxidant and MDA after the 90th and 163rd days of Cd treatment? To compare and make an accurate conclusion the same age of plant material should be used. Authors should also provide the exact age (days) of the material used.

The original plants were 3-month-old seedlings, 90-day-old seedlings.

51. Authors should stipulate how the different plant organs were dried (line 175).

The fresh plant samples were killed in a drying oven at 105℃ for 30min, then dried at 60℃ until constant weight, then the dried plant samples were crushed and passed through a 100-mesh nylon sieve, and then the dried plant samples were put into a microwave digestion tube, and 1 drop of purified water, 3 ml of nitric acid, and 1 ml of hydrofluoric acid were added sequentially.

Modified.

52. Rephrase lines 177-178.

Modified.

53. What is an acid-depleting apparatus? And what is the purpose of it in this assay? (lines 179-180).

Acid catcher, also called acid catcher electric heating plate, sample processor and ablator, is an aluminum alloy heating plate with the surface of Teflon fluoroplastic anti-corrosion and non-stick treatment.Measurement of heavy metals such as lead, cadmium, chromium, mercury, arsenic, zinc, selenium, manganese, and trace elements in food, pharmaceuticals, and dairy products.Determination of heavy metals and trace elements in soil by ablation .

54. Authors should rephrase lines 181 to 183.

Modified.

55. Authors should mention what solution was used as the blank control in line 184, for reproducibility purposes.

Modified.

56. Once again why was after 60 days used for cDNA construction? What exact day plant material was used? Mentioning after 60 days could mean either 90 days or 163 days.

The reason for selecting 60 days of Cd-stressed S. aculeatissimum for cDNA construction was that the Cd content of roots, stems, and leaves at 60 days was determined.

57. Did the authors mean ‘from the whole pot’ in line 193?

I'm sorry, I don't understand.

58. Were the plant organs of the same treatment all merged into one tube (lines 195-197). Authors should be clear in their entire methodology section.

Yes.Modified.

59. The subheading 2.8 does not match the description of the methodology there under lines 192-197.

Lines 192-197 describe the sampling method for sequencing.

60. Authors should mention whether they followed the manufacturer’s instructions when using the Total Plant RNA Extraction Kit (lines 199-200).

Modified.

61. In line 212, FPKM is the first mention, thus authors should write out the full acronym at first mention.

Modified.

62. Lines 222-224 are fragmented.

Modified.

63. Authors should clarify whether 5 primers were synthesized for each, or how was the distribution of synthesized primers (lines 231-232).

3 pairs of primers were synthesized.

64. Lines 234-239 explain the RNA extraction method. Should this method not have been explained in an earlier section (section 2.9)?

Both sequencing and real qrt-pcr real-time fluorescence validation require total RNA extraction, so the authors state this 2 separate times.

65. Italicize ‘S.aculeatissimum’ in line 247

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66. Authors should be specific to their investigation. Replace ‘heavy metal’ in line 249 with ‘Cd’.

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67. Throughout the manuscript authors should pay attention to the way they outline their treatment groups and the phrasing thereof.

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68. Why did the authors not consider investigating the phenotype of the rooting system? The authors spoke about the rooting system being the main entry point serving its importance in Cd uptake as well as authors investigated the rooting material in all other assays within the investigation (line 250).

Plant soil cultivation is not conducive to observing phenotypes.

69. Lines 250-251, the authors perfectly elucidated the number of treatment groups (6 different Cd concentrations). Authors should thus correct all the other mentions of 7 Cd-treatment groups.

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70. Correct the diagonal line in both Table 2 and Table 3.

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71. The alignment of the entire Cd6 row in Table 2 and Table 3 needs to be correct.

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72. Clear descriptions in the first cell in both Table 2 and Table 3 should be provided.

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73. Why is the plant growth only measured up to 60 days? Some of the other assays are conducted on much more mature plants. Where are the measurements for those days (days 90 to 163)?

When no significant leaf yellowing was seen on day 60, plant height and stem thickness measurements were not continued. Sequencing on day 60 was selected to correspond to the cadmium content of plants stressed on the same day, in response to the up- and down-regulation of the screened genes. Day 90 was chosen to measure plant physiological indicators because all treatment groups resulted in a shift from nutrient growth to reproductive reproduction.

74. Information about Notes of tables above line 281 is not for the table but for the Figure. Thus correct ‘The results in the Table’ to ‘The results in the Figure’.

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75. Stem thickness in line 255 is not shown in the results.

I'm sorry, I don't understand.

76. This is not true for Cd1, Cd4, Cd5, and Cd6 (line 258).

I'm sorry, I don't understand.

77. Authors should be consistent with what they are referring to when mentioning the result regarding the stem morphology. The Table indicates Stem length, whereas in the text the authors keep inter-changing stem length and stem height (line 259).

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78. Check alignment in each table in the entire manuscript.

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79. Authors should rephrase lines 289-291. Change ‘The variation of S. aculeatissimum content’ because authors are testing Cd content in S. aculeatissimum.

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80. All sub-images in Figure 3 have a labeled scale except Figure 3B. Please correct this.

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81. Authors should increase the font of axis labels in Figure 3, to make it more legible.

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82. The labeling for the control group should be kept consistent throughout the manuscript. The authors depicted the control group as CK however, in Figure 3, the control group is written as Cd0. To avoid confusion authors should change this Cd0 to CK.

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83. In Figure 3C the last bar for Cd6 is cut off.

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84. In Figure 3C, the authors represent roots, stems, leaves, unripe fruit, and ripe fruits, however, in the abstract when referring to this section of results, the author also mentioned mature fruit which is not presented here. Authors should consider either adding this result in the result section or removing the mention of the mature fruit.

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85. Throughout the manuscript the authors refer to the enrichment coefficient, however in Table 5 it is referred to as the Bioconcentration factor. Authors should please be consistent in the terminology used, whether in text or Figures and Tables.

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86. When referring to the transport coefficient in Section 3.2.2 are the authors referring to the translocation of Cd or what are the authors referring to?

Authors could refer to the translocation of Cd.

87. As mentioned previously, authors should review the Figures and the font sizes of the axis to make them more legible.

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88. Authors should also keep the Figures' labelling layout consistent throughout the manuscript. Figure 3 The Figure did not have a heading and the y-axis had more information whereas in Figure 4 the Figures have headings and the y-axis labelling only consists of SI units.

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89. Figure 4 legend should indicate on what day these measurements were taken. The figure legend should also indicate what plant organ these results were extrapolated from.

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90. The authors mention in section 3.4 that the average GC content was 48.38 %, however, Table 6 depicts a different value (46.90).

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91. The author also keeps mentioning ‘three samples’ however in the Tables only two samples are presented) (Ck and Cd6).

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92. Authors should correct Table 7 so that it is a stand-alone Table from Table 6.

I'm sorry, I don't understand.

93. Once again Authors should revise the font for Figures.

Modified.table 8 and table 9.

94. Figure 5 legend only mentions the Volcano plot, however, the Figure is of both a Volcano plot and a Scatter plot.

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95. The authors mention the 5 highest DEG groups however, what the authors listed does not correlate to the Figure. The 5 highest DEG groups appear to be Global and overview maps, Signal transduction, immune system, carbohydrate metabolism, and amino acid metabolism (Lines 364-365).

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96. Where are the results that correlate with section 3.8? where is this information extrapolated from?

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In Table 10.

97. What do the authors mean when saying ‘results of S. aculeatissimum content’ in lines 405-406? What content is being referred to? The authors should be specific.

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98. Authors mention ‘SaACs4’ in line 408, however, this gene was not represented in the figure.

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99. Is position the correct term to use? Should authors not refer to the x-axis as plant organs?

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100. Provide a reference for the first statement in the discussion (lines 411-413).

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101. What do the authors mean when they say’ change in height and stem height of S. aculeatissimum was not the same’ in line 417?

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102. The authors once again refer to stem thickness in line 423. Are the authors correlating stem height, stem length, and stem thickness? Because Stem thickness is not presented in the result section.

By mentioning stem diameter in the discussion, the authors are expressing that plant stem thickness is affected by cadmium stress.

103. In lines 428-430, the authors mention that increased Cd promoted leaf number, which was the case in their investigation, compared to their control. However, the authors go further in saying that with prolonged stress time, an inhibitory effect was seen on the leaves, which was not the case. The majority of the Cd treatments from day 15- day 90 illustrated more leaves than the control group (Figure 2).

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104. Rephrase lines 445-447, as the first half of the sentence makes it sound like MDA decreases with an increase in Cd concentration, which is not the case.

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105. In lines 452-453, the authors mention the MDA content of the roots and leaves, however, the only result focused on MDA (Figure 4 A) does not stipulate which plant organ it is conducted on. Please correct this.

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106. The authors mention the studies in lines 447-452 however do not compare this study with their current results.

In this study, we found that the MDA content of S. aculeatissimum decreased with increasing Cd stress concentration after reaching a peak in the Cd5 group. In studies on potatoes.

107. Lines 455-457, are the authors referring to their current investigation? Or are the authors referring to another study that was conducted?

The authors are referring to another study.

108. The conclusion in lines 465-468 cannot be made without the supporting evidence. The authors did not conduct hydrogen peroxide determination so authors cannot say that hydrogen peroxide was cleared by CAT. The same can be said with the statement in line 465, no ROS determination assays were conducted thus authors cannot conclude that there was excessive ROS produced they can only postulate this.

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109. Once again Figure 4 did not stipulate the organ that was used for this antioxidant determination, however in lines 473-474 the authors mentioned leaves.

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110. The authors cannot make conclusive remarks about antioxidants and their activity over prolonged periods of Cd stress as they did not include the MDA measurements at 163 days. In this investigation, authors could only extrapolate conclusive remarks from day 90.

Yes.I can only prove that it was the 90th day that I began to extrapolate the concluding observations.

111. In line 489 authors did not previously mention these acronyms. Thus, it is suggested that the authors write out the full words and then include the acronyms.

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112. Provide references for lines 491-493.

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113. In lines 518-519, there is repetition at the beginning and end of this sentence.

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114. The authors mentioned the KEGG results, and the genes however did not give a brief explanation as to the importance of these genes about stress. Were they involved in Cd stress response or Cd stress tolerance?

Also involved in cadmium stress response and cadmium stress tolerance.

115. The authors mention ‘The main antioxidant systems involved include antioxidant enzymes, glutathione 521 transferase, oleuropein lactone biosynthesis, and hydrolase activity’ in lines 521-522. Under what conditions are these antioxidants the main antioxidant systems? Furthermore, what is the purpose of mentioning this if none of these antioxidants were investigated in the current study? Why did the authors not investigate these antioxidants if they are referred to as the main antioxidant systems?

The authors selected metabolic pathways with a high number of differential genes only through KEGG database annotation and GO database annotation, and did not mine stress genes from metabolic pathways related to the antioxidant system, because the authors chose to screen from the significance of differentially expressed genes, and combined with the review of the relevant literature on heavy metals, they screened out five genes for late qRT-PCR validation.

116. Paragraphs 521-530, refer to genes related to antioxidant systems, however, nowhere in the manuscript do the authors mention or present results elucidating or identifying any genes involved in the antioxidant systems. The only results provided for antioxidants refer to the activity of antioxidant enzymes.

The authors selected metabolic pathways with a high number of differential genes only through KEGG database annotation and GO database annotation, and did not mine stress genes from metabolic pathways related to antioxidant system.

117. In lines 547-554, authors should use the literature they mentioned (lines 547-552) to conclude or explain their results in lines 552-555.

I'm sorry, I don't understand.

118. Correct the font of ‘Nightshade’ in line 575.

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Author Response File: Author Response.pdf