Lead (Pb)-Induced Oxidative Stress Alters the Morphological and Physio-Biochemical Properties of Rice (Oryza sativa L.)

Round 1

Reviewer 1 Report

Authors answered my queries. However, I feel that the present form of manuscript still have reference issues. Some references are listed twice in references. for example 12-24; 27-56; 35-37. Authors should check all of them to fix this duplication issue.

Author Response

Lead (Pb)-Induced Oxidative Stress Alters the Morphological and Physio-Biochemical Properties of Rice (Oryza sativa L.)

Manuscript ID: agronomy-1094475

Point by point reply to comments of reviewers

We are thankful to the editorial team and anonymous reviewers for their time given to this manuscript. We appreciate their comments, and are happy to share that most of the comments are addressed and have substantially improved the quality of the manuscript. We would like to specify that all changes in the manuscript were highlighted yellow. The text highlighted in green are from the previous round of revision. No track change was applied to the manuscript. We hope that the manuscript in the present form will be suitable for publication in the journal.

Reviewer 1
Authors answered my queries. However, I feel that the present form of manuscript still have reference issues. Some references are listed twice in references. for example 12-24; 27-56; 35-37. Authors should check all of them to fix this duplication issue.	We would like to apologize for the inconvenience. We have removed the duplicated reference from the revised manuscript as suggested by the reviewer.

Reviewer 2 Report

The manuscript describes an assessment of Pb-induced oxidative stress in rice examined with morphological and biochemical endpoints. Active mining and industrial developement in the last century resulted in global soil pollution with toxic heavy metals. One way to find solution of the problem is the choice of tolerant cultivars of agricultural plants. Suppose estimations of differences in Pb accumulation in rice grains among cultivars would be an extension of presented work.

Author Response

Lead (Pb)-Induced Oxidative Stress Alters the Morphological and Physio-Biochemical Properties of Rice (Oryza sativa L.)

Manuscript ID: agronomy-1073491

Point by point reply to comments of reviewers

We are thankful to the editorial team and anonymous reviewers for their time given to this manuscript. We appreciate their comments, and are happy to share that most of the comments are addressed and have substantially improved the quality of the manuscript. We would like to specify that all changes in the manuscript were highlighted yellow. The text highlighted in green are from the previous round of revision. No track change was applied to the manuscript. We hope that the manuscript in the present form will be suitable for publication in the journal.

Reviewer 2

The manuscript describes an assessment of Pb-induced oxidative stress in rice examined with morphological and biochemical endpoints. Active mining and industrial development in the last century resulted in global soil pollution with toxic heavy metals. One way to find solution of the problem is the choice of tolerant cultivars of agricultural plants. Suppose estimations of differences in Pb accumulation in rice grains among cultivars would be an extension of presented work.

The authors are thankful to the worthy reviewer for the valuable and constructive comments and suggestions, which have substantially helped us improve the manuscripts.

Reviewer 3 Report

The study touches several aspects related to Pb toxicity, however, the authors should state what is the novelty of the study and what new knowledge and concepts will add to the scientific literature. The correlation between the production of antioxidant enzymes and HMs as well as growth reduction and chlorophyll reduction have been already widely covered by the scientific literature. The presented study has serious methodological gaps (for examples it lacks elemental analysis). The English language is imprecise and needs revision.

 

Overall comments

There are several grammar errors, the whole manuscript would benefit from native-speaker revision. Just to cite some corrections that can be made: at line 64 “Over-accumulation”. At line 85 this sentence need revision: “The current European registration, regulation, and authorization of chemical substances (REACH) regulation”. At line 99 “were”. At line 124 “post-harvest”. At line 443 “enzymatic  activity”

Introduction section

The manuscript completely lacks a part in which the aims of the study are explained, apparently there are no scientific questions that guided this study.

A lot of imprecisions can be found in the method sections, for example at line 168 the authors just said “using a spectrophotometer” without specifying brand and model. Similarly brands and purity of the used reagents were never cited in the text.

In many cases the authors wrote “ as described by [36]” instead of writing by “Sirhindi et al. [36]”, this would make the sentence more fluent. Moreover, not all citations are reported as numbers in brackets as in line 172.

A “data analysis” paragraph must be present, details about the statistics used to validate the study results must be provided.

Results section

There are no values reported in the text, the reader would benefit from average values of the discussed paraments reported in text. It is not easy to follow the result presentation while constantly looking at the figures in search of the desired data. Moreover, graphs in Figure 1 are rather small.

There is no legend in Figure 2

Discussion

As the author stated in the first part of the discussion Pb is strongly bound to organic matter in soil, and yet there are no information about the organic matter content of medium used for this experiment (Doobaena plus). To avoid biases, quartz sand is usually choosen as growing medium.  If the organic matter content of the utilized medium is high, the added Pb might have been mostly unavailable to plants, and the observed toxicity effects on rice seedling might have been mitigated. To overcome this problem, I suggest to report the media composition in “material and method”, but also I would strongly recommend an elemental analysis of samples.

The effect of Pb on plant metabolism and growth, must be supported by an elemental quantification of at least of Pb inside plant shoots.

The quantification of Pb inside plants shoot could reveal that the more Pb-tolerant varieties are instead Pb-excluder and they just do no absorb Pb like the others varieties.

 

 

Author Response

Lead (Pb)-Induced Oxidative Stress Alters the Morphological and Physio-Biochemical Properties of Rice (Oryza sativa L.)

Manuscript ID: agronomy-1073491

Point by point reply to comments of reviewers

We are thankful to the editorial team and anonymous reviewers for their time given to this manuscript. We appreciate their comments, and are happy to share that most of the comments are addressed and have substantially improved the quality of the manuscript. We would like to specify that all changes in the manuscript were highlighted yellow. The text highlighted in green are from the previous round of revision. No track change was applied to the manuscript. We hope that the manuscript in the present form will be suitable for publication in the journal.

Reviewer 3
The study touches several aspects related to Pb toxicity, however, the authors should state what is the novelty of the study and what new knowledge and concepts will add to the scientific literature.   The correlation between the production of antioxidant enzymes and HMs as well as growth reduction and chlorophyll reduction have been already widely covered by the scientific literature.   The presented study has serious methodological gaps (for examples it lacks elemental analysis). The English language is imprecise and needs revision.	The authors appreciate the concern raised by the reviewer. We apologize for any inconvenience caused by a potential unexplained objectives and major contribution of the current study to the general audience and existing literature in the field of study. In principles, authors agree with the point raised by the reviewer, and this could be true for any other abiotic or biotic stress inducer. The general aspects of the abiotic stress response mechanism in plants have been established, which include activation of antioxidant systems (enzymatic and non-enzymatic), the change in the physiological processes and biochemical reactions. Usually, these metabolic components are take separately, and in a few cases, their interactions are investigated, and in some situations, their interplay is not well elucidated due to many reasons, including the genetic variability of the materials used coupled with the level of the stress and the concentration of the stress inducer as well as the time of exposure., among other others. The reviewer is also questioning the contribution of the present study to the scientific literature. Here, we would like to emphasize that many enzymatic antioxidants play a positive role in alleviating the oxidative stress induced by the imbalance between reactive oxygen species (ROS) or reactive nitrogen species (RNS) and antioxidants. The common ones include catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), and superoxide dismutate (SOD). In many cases, the action of these antioxidant enzymes is evaluated independently. But, here we have challenged to analyze and broadly discuss their possible interplay during stress conditions, here in the case of lead (Pb) treatment. From the observed results, it is suggested that the observed tolerance of Pb in some of the tested rice cultivars may be attributed to the combinational action of antioxidant enzymes, coupled with some physiological compounds such as soluble sugars (Sucrose which accumulated abundantly, compared to Glucose and Fructose), chlorophylls and proline. For this reason, we have suggested that the observed Pb tolerance in particular Tunnae and Mashkab would be attributed to a synergetic action of both enzymatic and non-enzymatic antioxidant systems. The soluble sugars are here identified as emerging biomarkers that be use when assessing the overall health status of the plant.
Overall comments
1.      There are several grammar errors, the whole manuscript would benefit from native-speaker revision. Just to cite some corrections that can be made: at line 64 “Over-accumulation”. At line 85 this sentence need revision: “The current European registration, regulation, and authorization of chemical substances (REACH) regulation”. At line 99 “were”. At line 124 “post-harvest”. At line 443 “enzymatic  activity”	Authors sincerely appreciate the concern raised by the reviewer in order to improve the quality of the manuscript. We have tried to review the manuscript for English and possible grammatical errors as suggested.
Introduction section
2.      The manuscript completely lacks a part in which the aims of the study are explained, apparently there are no scientific questions that guided this study	Lines 89-94: we have included the following statement: “In addition to the enzymatic antioxidant system and their interplay, we assessed the possible involvement of the soluble sugars in the adaptive response mechanism towards lead (Pb)-induced oxidative stress by investigating the changes in the sucrose, glucose and fructose contents in indica and japonica rice varieties originated from different geographic locations.”
3.      A lot of imprecisions can be found in the method sections, for example at line 168 the authors just said “using a spectrophotometer” without specifying brand and model. Similarly brands and purity of the used reagents were never cited in the text.	The authors appreciate the concern raised by the reviewer. We would like apologize for the inconvenience, and we have included the required information as suggested.
4.      In many cases the authors wrote “as described by [36]” instead of writing by “Sirhindi et al. [36]”, this would make the sentence more fluent. Moreover, not all citations are reported as numbers in brackets as in line 172.	We appreciate the suggestion and have modified accordingly. But we think that it is acceptable to write “as described earlier”
5.      A “data analysis” paragraph must be present, details about the statistics used to validate the study results must be provided.	We would like to apologize for the inconvenience. We have included a subsection describing the statistical analysis performed as recommended.
Results section
6.      There are no values reported in the text, the reader would benefit from average values of the discussed parameters reported in text. It is not easy to follow the result presentation while constantly looking at the figures in search of the desired data.   Moreover, graphs in Figure 1 are rather small.	The authors are thankful to the reviewer for his valuable suggestions that helped us improve the manuscript. We have included the estimation of the changes in the phenotypic response and physio-biochemical properties due to Pb treatment, in percentage and fold change respectively. The authors understand the concern raised by the reviewer, but we would like to indicate that it is always challenging to fit many panels in a single figure to allow the readers to have an overview of the recorded phenotypes. We could not increase the size of all panels on the left of the figure beyond the page limits.
7.      There is no legend in Figure 2	We would like to apologize for the inconvenience. We have included the legend in the figure 2
8.      Discussion
9.      As the author stated in the first part of the discussion Pb is strongly bound to organic matter in soil, and yet there are no information about the organic matter content of medium used for this experiment (Doobaena plus). To avoid biases, quartz sand is usually chosen as growing medium.  If the organic matter content of the utilized medium is high, the added Pb might have been mostly unavailable to plants, and the observed toxicity effects on rice seedling might have been mitigated. To overcome this problem, I suggest to report the media composition in “material and method”, but also I would strongly recommend an elemental analysis of samples.	The authors appreciate the concern raised by the reviewer. We would like to indicate, on the one hand, that the soil used to conduct the experiments was a commercial soil purchased from an accredited manufacturer specialized in preparing soil for growing rice. The use of the soil was done following the manufacturer’s instructions, and the soil composition could not be disclosed by the manufacturer for specific and evident reasons. A similar case is observed for Hoagland nutrients (salt and calcium nitrate) reagents, of which the detail composition is not disclosed, but is said to contain basic nutrients necessary for growing plants. On the other hand, the use of control treatments where no exogenous lead (Pb) was applied to assess the effects of the applied Pb concentrations on different rice growth related parameters and yield components, coupled with the changes in the physiological process and the biochemical reactions. Nevertheless, it is evident that the gradient lead concentrations had a significant effect on the yield components and physio-biochemical properties of rice in a variety dependent manner. But, these antioxidants were differentially affected, and based on the accumulation pattern in Pb tolerant and sensitive varieties. When taken individually, CAT, POD, PPO, and SOD may not provide the required level of tolerance to maintain a balanced reduction-oxidation status. From this perspective, it is suggested that a combinational actions of enzymatic and non-enzymatic antioxidant  have the potential to enhance the level of tolerance towards Pb stress in rice, rather than individual actions.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

In this article, authors exposed 5 rice cultivars to Pb and investigated morphological and Physio-biochemical response in those cultivars. Authors have answered all the queries and significantly improved the manuscript. 

Author Response

The authors would like to thank the reviewer for his precious time given to revised our manuscript, and the quality of the manuscript has been improved substantially.

Reviewer 3 Report

The article has been widely improved aspecially in the section results, a defined aim of the study has been stated and the overall scope of the study in snoe clearer.

I still don't find  a paragraph in which data analisys is clearly explained, for  example it's not stated whether the differences among groups were tested using ANOVA, or other, and which post-hoc testes were then applied. 

Please add these information in a dedicated paragraph in "materials and methods"

Author Response

 The authors would like to apologize for the inconvenience. Therefore, we have included a section describing the statistical analysis performed to evaluate the significance of the data within and between groups as follows:

Lines 208-2013:

2.7. Statistical Analysis

All the experiments were done using CRD design. The data were collected in triplicates and analyzed statistically with GraphPad Prism software (Version 7.00, 1992-2016 GraphPad). The analysis of variance (ANOVA) for Completely Randomized Design was performed, and the Turkey’s multiple comparison was employed at a significance level of 0.05.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

In this article, authors exposed 5 rice cultivars to Pb and investigated morphological and Physio-biochemical response in those cultivars.

My specific comments are;

Authors should check the Pb levels in roots, otherwise all the correlation of all the data can be misleading.

References are inappropriate. For examples Line 299-303;, ref#45 is for brassica napus and it is not well suited over here. Secondly ref#46 is not from Ali et al; . This is very bad, because we don’t know whether all the references in the manuscript were correctly cited. Moreover, ref#47 I don’t understand how it can be cited here? I wonder if it is deliberate attempt to cite certain references from the group or from co-authors?

Authors should add background and rational of this study in the abstract.

What was the criteria and rational to choose these varieties? Why Korea and Iraq?

How authors decide to use 0.6mM and 1.2mM of Pb? Any evidence from literature? It does not seems to cause enough stress consitions.

Figure 1 A and 2A-D, It seems that authors deleted the name below of each graph, but it still appears. Better to fix this issue.

Figure 2D in Mashkab, why the SOD levels are lower at higher 0.6mM but not changed at 1.2mM? its quite strange.

Figure 3 and 4 (A-B), because all the graphs are not in one panel, it is better to add varieties name below the graphs.

Discussion should be improved. It is very superficial and no deep understanding shown in the discussion.

Discussion section 4.1; It is essentially the repetition of results. Also cited references are not essentially on rice, where I believe one can find plenty of information. Similarly section 4.2; 313-322 and so on..

If authors explanation is valid (line 308-312), How author explain the levels of Antioxidants in addition to plant morphology and phenotype from Fig 1?

Line 332-334; Authors should discuss why SOD and O2- levels are not systematically matched?

Reviewer 2 Report

The manuscript presented is relate to assessment of Pb-induced oxidative stress in rice examined with morphological and biochemical endpoints. The article is interesting from the point of view of use of different rice cultivars in case of soil contamination with heavy metals. However, there are some questions to be answered.

1. Authors have chosen O₂^•- examination as a measure of reactive oxygen species caused with Pb oxidative stress. Unfortunately, no explanations were presented why this one of oxygen species had being chosen among others. According to data on changes in antioxidant enzyme levels resulted from Pb stress – catalase has the most pronounced and stable effect. So why O₂^•- and not hydrogen peroxide and other ROS was measured?
2. Rice is valuable for it grains. Does effects on yield and Pb accumulation in grain were assessed in cultivars studied?

 

Minor comments

1. Lines 207-209: …examples of enzymatic and non-enzymatic antioxidants includes only enzymes. It would be better to add some non-enzymatic antioxidants too or rebuild the sentence.
2. Line 212-214: “high CAT activity was recorded in Ilmi, Yasmen, and Mashkab under 0.6 mM Pb; meanwhile, Tunnae and Amber Barka showed a similar CAT activity at 0.6 mM Pb treatment." It is not quite clear to what was CAT activity at 0.6 mM Pb treatment was similar in Tunnae and Amber Barka.
3. Figure 2. The figure caption is incorrect. Under the letter E – there is superoxide ion level and F – leakage level.
4. Line 282: to easier understanding it is better to transfer ppm to mM in brackets.
5. Line 340: excess point after reference.
6. Line 346-347: is that all ok with contribute”?