Genotype-Dependent Differences between Cereals in Response to Manganese Excess in the Environment
, Apolonia Sieprawska 1,*, Elżbieta Bednarska-Kozakiewicz 2, Katarzyna Gawrońska 1, Andrzej Kornaś 1 and Anna Telk 3Round 1
Reviewer 1 Report
I already reviewed this article and gave some corrections to the authors.
It seems like they addressed all of the corrections and I think paper can be published as it is.
Author Response
Response to Reviewer 1 Comments
Thank you for accepting all corrections in our revised manuscript.
Reviewer 2 Report
Report on agronomy-763756-v1
It is quite obvious that the authors have worked over their manuscript and followed most of suggestions. Unfortunately, in newly added parts some more corrections are needed, but this should not take more than a few hours.
Line 16: ... and then to 16 media on which... (is it this what the authors want to say?)
18: Under Mn-stress a decrease in fresh weight of plants was observed, also differences in water content in first leaves, an increase in... activity, and a significant rise in catalase (CA) was only characteristic for barley. (Suggestion!)
21: copper, zinc superoxide dismutase (no italics!)
22 ff: The increase in proline content, depending on a balance between pyrroline-5-carboxylate synthase (P5CS), ornithine-d-aminotransferase (OAT) and proline dehydrogenases (PHD) activities, indicated osmotic disorders in all plants and differentiated the studied cereals. (Suggestion)
45 ff: Niu et al. [10] reported Mn soil contents in China in the range of 40-1681 mg / kg, and Antibachi et al. [11] measured values ranging from 685-1307 mg / kg in soil from central Greece. (Suggestion!)
60: Presumably, a ROS surplus may lead to... (or: may be leading to...)
94: ... were received from the Polish Plant Breeding...
114: AGAIN: What is the meaning of "Mn (55)"? Do the authors want to say Mn2+?
137: Delete the second "according to"
140: ... were loaded on a 12% gel.
154: Ornithine-D-aminotransferase (perhaps a smaller letter size for the "D")
192 ff: All cultivars responded to a higher Mn dose with a drop in their water content, with the effect being particularly strong in the sensitive plants (about 69 - 74%). All cultivars indicated as tolerant in our earlier work [citation?] were characterized by a statistically significant higher mass than that of sensitive ones, both in control conditions and under Mn treatment. (Suggestion! But as it is, it is not easy to understand)
200 ff: In the sensitive objects (plants?), a higher Mn dose nearly doubled the enzyme activity in comparison with the control.
224: ... that the salicylic acid (SA) concentration...
Page 10, line 19: ... at 10 than at 5 mM MnCl2.
Figure 4 A and line 27: Once again: [µM mg-1protein] is not a correct dimension! REPLACE by: µmol mg-1 protein, also in the diagram.
34: In parenchymal cells, numerous...
52 ff: Already Izaguirre-Mayoral and Sinclair [51] confirmed the role of interspecific and phenotypic variations in crops after experimental overexposure to Mn. (Suggestion)
134 ff: The exception is the oat Siwek, where the highest metal accumulation could cause irreversible metabolic dysfunctions (including reduction of enzyme activity), which particularly resulted in fresh mass reduction under Mn stress conditions.
149: ... in the sensitive oat cultivar
178: Ann. Bot.2015,
197: J. Plant. Physiol.
Page 17, line 27: New Phytol.
250: J. Plant Physiol.
260 ff: Lück H. In: "Methods of Enzymatic Analysis." 1st ed. (Bergmeyer, H.U., editor). Vol. 1. (Verlag Chemie G.m.b.H., Weinheim/Bergstr. (Germany) and Academic Press; New York and London: 1963. pp. 895–897. (A least the part that is underlined should be quoted!)
273: Plant Soil
282: 24-epibrassinolide
Author Response
Response to Reviewer 2 Comments
Thank you for Your comments that used improve our article. All your suggestions have been considered in the revised version and marked as “red”.
Report on agronomy-763756-v1
It is quite obvious that the authors have worked over their manuscript and followed most of suggestions. Unfortunately, in newly added parts some more corrections are needed, but this should not take more than a few hours.
Ad. Line 16: ... and then to 16 media on which... (is it this what the authors want to say?)
We corrected this sentence as:
“Manganese ions (MnCl2) at 5 and 10 mM were applied to the grains and then to the media on which the plants grew until they developed their first leaf.”
Ad. 18: Under Mn-stress a decrease in fresh weight of plants was observed, also differences in water content in first leaves, an increase in... activity, and a significant rise in catalase (CA) was only characteristic for barley. (Suggestion!)
According to Your suggestion we corrected the sentence as:
“Under Mn-stress a decrease in fresh weight of plants was observed, also differences in water content in first leaves, an increase in superoxide dismutases (SOD) and peroxidases (POX) activity, and a significant rise in catalase (CAT) was only characteristic for barley.”
Ad. 21: copper, zinc superoxide dismutase (no italics!)
According to Your suggestion we checked and corrected as:
“Increasing Mn concentration resulted in enhancing of manganese superoxide dismutase (Mn-SOD) and copper, zinc superoxide dismutase (Cu,Zn-SOD) bands intensity.”
Ad. 22 ff: The increase in proline content, depending on a balance between pyrroline-5-carboxylate synthase (P5CS), ornithine-d-aminotransferase (OAT) and proline dehydrogenases (PHD) activities, indicated osmotic disorders in all plants and differentiated the studied cereals. (Suggestion)
According to Your suggestion we corrected this sentence as:
“The increase in proline content, depending on a balance between pyrroline-5-carboxylate synthase (P5CS), ornithine-d-aminotransferase (OAT) and proline dehydrogenases (PHD) activities, indicated osmotic disorders in all plants and differentiated the studied cereals.”
Ad. 45 ff: Niu et al. [10] reported Mn soil contents in China in the range of 40-1681 mg / kg, and Antibachi et al. [11] measured values ranging from 685-1307 mg / kg in soil from central Greece. (Suggestion!)
According to Your suggestion we corrected the sentences:
“Niu et al. [10] reported Mn soil contents in China in the range of 40-1681 mg / kg, and Antibachi et al. [11] measured values ranging from 685-1307 mg / kg in soil from central Greece.”
Ad. 60: Presumably, a ROS surplus may lead to... (or: may be leading to...)
According to Your suggestion we corrected the sentence as:
“Presumably, a ROS surplus may lead to the activation of protective antioxidants of enzymatic and non-enzymatic origin [22].”
Ad. 94: ... were received from the Polish Plant Breeding...
According to Your suggestion we corrected the text as:
“…were received from the Polish Plant Breeding Stations Radzików and Strzelce.”
Ad. 114: AGAIN: What is the meaning of "Mn (55)"? Do the authors want to say Mn2+?
The 55Mn (100% stable) isotope was analyzed, i.e. the one that occurs only in nature. All others are obtained artificially and are radioactive.
We corrected the sentence as:
“55Mn was analyzed by ICP-MS spectrometry (Elan DRC-e, Perkin Elmer, Shelton, USA), according the protocol described in details by Tobiasz et al. [37].”
Ad. 137: Delete the second "according to"
According to Your suggestion we corrected the sentence as:
“Protein content was quantified according to Bradford [41].”
Ad. 140: ... were loaded on a 12% gel.
We corrected the sentence as:
“Samples containing 9 µg of protein were loaded on a 12% gel.”
Ad. 154: Ornithine-D-aminotransferase (perhaps a smaller letter size for the "D")
For this enzyme three forms can be used: ornithine d-aminotransferase (but a smaller letter size for the "D"), ornithine δ-aminotransferase or 5-aminotransferase.
Ad. 192 ff: All cultivars responded to a higher Mn dose with a drop in their water content, with the effect being particularly strong in the sensitive plants (about 69 - 74%). All cultivars indicated as tolerant in our earlier work [citation?] were characterized by a statistically significant higher mass than that of sensitive ones, both in control conditions and under Mn treatment. (Suggestion! But as it is, it is not easy to understand)
According to your suggestion we corrected the sentences as:
“All cultivars responded to a higher Mn dose with a drop in their water content, with the effect being particularly strong in the sensitive plants (about 69 - 74%). All cultivars indicated as tolerant in our earlier works [34, 35, 36] were characterized by a statistically significant higher mass than that of sensitive ones, both in control conditions and under Mn treatment.”
Ad. 200 ff: In the sensitive objects (plants?), a higher Mn dose nearly doubled the enzyme activity in comparison with the control.
According to Your suggestion we corrected the sentence as:
“In the sensitive plants, a higher Mn dose nearly doubled the enzyme activity in comparison with the control.”
Ad. 224: ... that the salicylic acid (SA) concentration...
According to Your suggestion we corrected the sentence.
Ad. Page 10, line 19: ... at 10 than at 5 mM MnCl2.
According to Your suggestion we corrected the sentence as:
“The only exception was cv. Siwek, where P5CS was slightly less active at 10 than at 5 mM MnCl2.”
Ad. Figure 4 A and line 27: Once again: [µM mg-1protein] is not a correct dimension! REPLACE by: µmol mg-1 protein, also in the diagram.
According to Your suggestion we corrected [µM mg-1protein] to µmol mg-1 protein also in the diagram.
Ad. 34: In parenchymal cells, numerous...
According to Your suggestion we corrected the sentence as:
“In parenchymal cells, numerous plastids (arrows) were visible.”
Ad. 52 ff: Already Izaguirre-Mayoral and Sinclair [51] confirmed the role of interspecific and phenotypic variations in crops after experimental overexposure to Mn. (Suggestion)
According to Your suggestion we corrected the sentence as:
“Already Izaguirre-Mayoral and Sinclair [51] confirmed the role of interspecific and phenotypic variations in crops after experimental overexposure to Mn.”
Ad. 134 ff: The exception is the oat Siwek, where the highest metal accumulation could cause irreversible metabolic dysfunctions (including reduction of enzyme activity), which particularly resulted in fresh mass reduction under Mn stress conditions.
According to Your suggestion we corrected the sentence as:
“The exception is the oat Siwek, where the highest metal accumulation could cause irreversible metabolic dysfunctions (including reduction of enzyme activity), which particularly resulted in fresh mass reduction under Mn stress conditions.”
Ad. 149: ... in the sensitive oat cultivar
According to Your suggestion we corrected the sentence as:
“Moreover, in the sensitive oat cultivar the higher dose of Mn significantly activated the enzyme responsible for proline degradation that could consequently weaken cellular protection in this cultivar.”
Ad. 178: Ann. Bot.2015,
We corrected the reference as:
“Fernando, D.R.; Lynch, J.P. Manganese phytotoxicity: new light on an old problem. Ann. Bot. 2015, 116, 313-319.”
Ad. 197: J. Plant. Physiol.
We corrected the reference as:
“Kurdziel, M.; Dłubacz, A.; Wesełucha-Birczyńska, A.; Filek, M.; Łabanowska, M. Stable radicals and biochemical compounds in embryos and endosperm of wheat grains differentiating sensitive and tolerant genotypes – EPR and Raman studies. J. Plant Physiol. 2015, 183, 95–107.”
Ad. Page 17, line 27: New Phytol.
We corrected the reference as:
“Kameli, A.; D. M. Lösel. Carbohydrates and water status in wheat plants under water stress. New Phytol. 1993, 125, 609-614.”
Ad. 250: J. Plant Physiol.
We corrected the reference as:
“Łabanowska, M.; Kurdziel, M.; Filek, M.; Wesełucha-Birczyńska, A. The impact of biochemical composition and nature of paramagnetic species in grains on stress tolerance of oat cultivars. J. Plant Physiol. 2016, 199, 52–66.”
Ad. 260 ff: Lück H. In: "Methods of Enzymatic Analysis." 1st ed. (Bergmeyer, H.U., editor). Vol. 1. (Verlag Chemie G.m.b.H., Weinheim/Bergstr. (Germany) and Academic Press; New York and London: 1963. pp. 895–897. (A least the part that is underlined should be quoted!)
Thank you for paying attention to the Lück reference, we corrected it’s as:
“Lück H. In: "Methods of Enzymatic Analysis." 1st ed. (Bergmeyer, H.U., editor). Vol. 1. (Verlag Chemie G.m.b.H., Weinheim/Bergstr. (Germany) and Academic Press; New York and London: 1963. pp. 895–897.”
Ad. 273: Plant Soil
We corrected the reference as:
“Bates, L.S.; Waldren, R.P.; Teare, I.D. Rapid determination of free proline for water-stress studies. Plant Soil 1973, 39, 205-207.”
Ad. 282: 24-epibrassinolide
We corrected the reference as:
“Janeczko, A.; Biesaga-Kościelniak, J.; Oklestkova, J.; Filek, M.; Dziurka, M.; Szarek-Łukaszewska, G.; Kościelniak, J. Role of 24-epibrassinolide in wheat production: Physiological effects and uptake. J. Agron. Crop Sci. 2010, 196, 311–321.”
Reviewer 3 Report
The authors well answered to my questions in the revised version.
Author Response
Response to Reviewer 2 Comments
Thank you for accepting all corrections in our revised manuscript.
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
The findings of this article are interesting, however, it needs extensive English editing before publication. It seems like Mn in cereals is not well studied, so the author`s findings are worth publishing. However; the manuscript should be edited intensively. Please see my comments below. For the improvement of grammar/ English, you can use online grammar tools to make sentences understandable. Some parts really do not make any sense. Using shorter sentences with less comma, more periods may help.
Abstract: This abstract is very badly written. The information in the abstract should be re-organized and should be written in a better way. Also, it should provide more information regarding to the findings of the study.
- The language should be improved. The abstract is informative but very badly written.
For instance:
In this study differences in response to Mn excess in the most popular cereals such as wheat, oat and barley, and their sensitive and tolerant cultivars were investigated.
Can be written as: In this study, different cultivars of popular cereals wheat, oat, and barley were investigated for their response to excessive Mn.
- Please provide long names for SOD/ POX.
- Do not provide this in the abstract.
Manganese ions (MnCl2) at 5 and 10 mM were applied to the grains and then to 16 the media on which the plants grew until they developed their first leaf. ICP MS.
This can be written as the following:
We applied 2 levels of Mn stress to different cereals and performed ICP MS aiming to understand the mechanism of manganese stress in susceptible and resistant cultivar. We observed x, y, z changes…
- What is the conclusion of this study? In the beginning, you mention that there is little know for Mn response. Did your study contribute to the understanding of manganese triggered stress? The last part of the abstract should mention this.
Introduction: Provided information is OK however, English writing is not good. There are sentences that are not understandable at all. Only, the last paragraph is good. The rest of it should be re-written.
- Authors can mention more about the problem of Mn stress world wide for cereals. What percentage of the worlds do soils suffer from excessive Mn?
- I seriously do not understand sentences in the first 3 sentences. Please fix this. Example below:
Increasing demand for cereals, which are currently one of the basic foods in the world, calls for genotypes resistant to), leads to disturbanes nutrient diffusion and mass flow in the soil consequently to change their changing climatic conditions.
Materials & Methods: Overall, it needs improvement. Some things should be described better.
- How do you know that these varieties are susceptible/ resistant to Mn? Please provide information for this. Did you screen them prior to your analysis? If there are papers, please cite them here.
- Is this wheat variety winter wheat? Spring wheat? Same with the barley and oat? Is the barley six-row or two-row? Please provide more information regarding the plant material and used varieties.
- Did you use any kind of experimental design for your greenhouse experiments? How did you organize your pots? Did you make sure that plants got the same amount of sun/water etc? The authors need to mention their experimental design. They did some statistical analysis, it is important to know the details regarding how the plant material was generated.
- Authors use very long sentences, and they are hard to understand. Please use shorter sentences. Separate them with a period, not with comma.
- For the manganese content analysis, did you use the entire tissue? In your figure, you mention 5 replications. Did you get 5 replications from different leaves? Or different sections of the same leaf? Please provide more detail.
- Did you identify proline from fresh leaves? For each of these analyses, please provide more information. Also, authors can group the analysis, they do not have to make a different section for each. My suggestion is the following:
2.1. Plant Material and Growth Condition
2.2. Determination of Manganese content via Biochemistry and Microscopy Assays
2.3. Antioxidant Enzyme Assays and Detection of Their Isoforms
2.4. Analysis of Proline Metabolism
2.5. Carbohydrate and Salicylic Acid Content
2.6. Statistical Analysis of Data
- If you used any specific package in SAS, please provide information.
- Mention the replications in your measurements not only in the figure caption but also in your materials/methods.
Results: This part is clear, minor edits are required.
- Figure 3: I do not think these bands are very obvious. If possible, it would be getting a better PAGE picture.
- Figure 2: When the graphs are nearby each other like this, it is very hard to read them. Please organize them in a way that they would be more visible.
Discussion: Overall, this part is OK. Maybe, authors may add some information about the significance of Mn stress in cereals. Is it a serious problem? If it is, Which parts of the world are more susceptible? It may help to make the point for why the authors conducted this research in the first place. Especially, it would be helpful for the conclusion.
Reviewer 2 Report
Report on manuscript agronomy-733153, entitled "Genotype-dependent differences between cereals in response to manganese excess in the environment" by Magdalena Skórka et al.
Manganese is a heavy metal that might cause problems for crops when cultivated on industrially contaminated soils, but in form of Mn2+it is an essential cation in plant metabolism, being for instance active in photosynthesis, more precisely in the water-splitting complex. Furthermore, many enzymes need bivalent cations such as Mg2+, which can be frequently replaced by Mn2+, at least in vitro.
A surplus in one (essential) element that needs to be absorbed from soil, frequently using the same transportation systems, will easily lead to a lack in other essential cations (Fe, Zn, Cu...).
Over the decades, many studies have been carried out on soil contamination by heavy metals (with some focus on Cd), and on their effects on plant growth and productivity. The list of citations in this manuscript is to some extent reflecting this, including some publications from the authors' laboratory. This also indicates some lack of novelty...
There are some problems with the manuscript that need to be corrected. This does not only concern some statements and how they are presented, but also some calculations. This is a real pity in view of the rather carefully prepared list of references.
Abstract: What do the authors try to say with this: Of the ions plant require for their development relatively little is known about mechanisms of manganese triggered stress. Could be shorter, for instance like: Relatively little is known about mechanisms of manganese triggered stress.
Several parts of the manuscript would profit of some more elegant writing or the choice of words within the immense English vocabulatory...
In the following it will be referred to line numbers, with questions and (linguistic) suggestions.
31 ff: An increasing demand for cereals, as one of the basic foods in the world, calls for genotypes that are resistant to various stress factors, leading for instance to disturbances in nutrient diffusion and mass flow in the soil. Furthermore, cereals need to adapt to changing climatic conditions such as a progressive global warming, with a limited amount of rainfall [1]. (Guess this is what the authors want to say... The original statement is completely screwed up!)
36: ... exceeding the plants' demand or: ... exceeding the demand of plants
43 ff: The importance of Mn in the cell functioning consists in its location in the active center of many enzymes [9], especially those that are involved in the regulation of redox properties being disturbed under stressful conditions and triggered by reactive oxygen species (ROS) [10].
51: ... dependencies were also observed in other plants than cereals (?)
Sieprawska et al. [18] suggested that Mn could accumulate in chloroplasts, and hence this would explain the significant effect as to modification of stress reactions associated with the generation of excess ROS in these organelles.
54 ff: Generally, a ROS surplus is leading to the activation of protective antioxidants of enzymatic and non-enzymatic origin [19]. (Suggestion!)
Superoxide dismutases (SODs) catalyze the disproportionation reaction of the superoxide anion radical (O2•−), and catalases (CAT) and peroxidases (POX) eliminate hydrogen peroxide.
Among non-enzymatic antioxidants, an increase in the synthesis of proline and sugars, also ensuring the stability of osmotic conditions, has been reported frequently [20].
61: .... under stress and its role in stabilizing the osmotic environment in plant cells, was...
62: However, the mechanisms of this osmoprotectant synthesis are not fully understood, especially in view of (the) plant sensitivity to stress. (Suggestion!)
65: ... via the ornithine pathway by action of...
67 ff: An enhanced content of carbohydrates, serving as a source of energy essential for physiological processes was reported for many stresses [24] , including heavy metals [25]. In fact, the synthesis of proline and carbohydrates has been suggested as a crucial element in plant stress survival strategy [26]. (Suggestion!)
73 ff: The stress sensitive and tolerant cultivars of three cereals most commonly used in agriculture and basis for many food products, i.e., wheat, barley and oat were studied. (Suggestion)
77: oat, with its grains...
88: ... were received for the Polish...
90 ff: From the International Center for Agricultural Research in the Dry Areas (ICARDA), and pedigree and seed source information: http://genbank.vurv.cz/genetic/resources/asp2/default_a.htm, respectively.
103: Manganese (55) was analyzed by... (Correct citation in this context? Or what is the meaning?)
124: Protein content was quantified according to...
143: Lütts et al. [42].
157: ... preceding the microscopic....
166: A technical question: Have those seeds been washed? The procedure of preparing seeds for this type of analysis should be explained in more detail, especially after incubation with Mn.
170: ... increased by 7 and 9 fold ...about 13 and 15 fold for...
173: .... of the first leaf was higher
174 ff: A higher Mn dose decreased the fresh weight especially in the sensitive cultivars: by about 5 mg/plant in Raweta and Maresi and about 7 mg/plant in Siwek, while in all tolerant cultivars by about 2 mg/plant only.
179: .... sensitive plants (or: plant varieties)
182: B - fresh weight [mg] ... (cf. Y-axis!)
190: was the lowest (one).
Figure 2C: The values for CAT are comparably low. A technical problem perhaps? Catalase it the most active enzyme in terms of molecules converted per sec.
Figure 3: Nice in-gel reactions for SOD. The authors refer to the Laemmli system in the "Methods section". Usually the system is cited through the use of SDS. Please, check!
211: ... that the salicylic acid (SA) concentration (Table 1)...
214 ff: An exception to this pattern was cv. CAM/B1, where a higher Mn concentration triggered a greater increase in SA than the lower one. In sensitive wheat and oat the content of SA decreased with increasing Mn accumulation. In Maresi some increase of SA was noted only at 5 mM MnCl2.
Table 1: When consulting the corresponding literature in the field, those values for carbohydrates must be mg/g (DW!). In reverse, when looking into the corresponding literature as to SA, those values should most possibly be reduced by up to 100 fold or even more, means mg should be µg!
Page 8, line 2: ... than in tolerant plants,
Figure 4 A: What exactly are the units of the Y-axis? µmoles per mg protein? µM is nothing but a concentration! B, C, D: Write only "protein" in the Y-axis - like in the legend.
45: Proplastids
(Usually, the accumulation of starch is a sign of high PS activity: Soluble carbohydrates exerting an osomotic pressure inside the (chloro-) plastids would be convered into starch that is not an osmoticum. Isn't there some contradiction in the description here, and later on in the discussion?)
64 ff: ... symptoms of Mn stress included changes of water relations in the leaves of all cultivars, suggesting the occurrence of...
66: ... despite the similar water potential of ...
72: ... affected the activity of...
74: ... even in control conditions (or: under control conditions)
This may suggest a higher activity of wheat...
80: The previously suggested largest sensitivity of oats to Mn... (give a citation already here!)
86 ff: ... (superoxide anion radical), was already reported for wheat leaves in numerous studies [18,53]. Therefore, the analysis of the activity of individual SOD-isoforms was performed for this cereal.
97 ff: See a remark above as to starch accumulation (in photosyntheticalle active leaves?)
104: ... was suggested earlier by ...
108: In the present study (here), the increase...
117: ... are usually considered as...
124 ff: A conclusion should really summarize the major observations and not repeat most parts of results and discussion. Cut be easily cut by 50%!
179: Styloanthes(in italics?) BMC Plant Biol.
232: J. Plant Physiol.
246: Anal. Biochem.
263: (?) J. Agron. Crop. Sci.
290: Plant Sci.
Reviewer 3 Report
In this study, the authors evaluated effects of manganese (Mn) excess using Mn tolerant and sensitive lines in wheat, oats, and barley. They measured Mn contents using ICP-MS and FW, investigated activities of SOD, POX, and CAT. They also provided salicylic acid, carbohydrates and starch levels. Furthermore, they investigated proline levels, activities of P5C5, OAT, and PHD. In addition, they observed structural changes of plastids and starch accumulation in leaf cells by Mn excess. Finally, they claimed that wheat is the most tolerant to Mn excess among cereals they tested. The paper contains very interesting information. However, the reviewer considers that some data collections and revisions are required for the conclusion.
Major points
1) The authors used Mn-tolerant and sensitive lines in wheat, oats, and barley in this study. However, they did not discuss the differences on Mn tolerance between different lines well. For example, the reviewer cannot understand the differences between Parabola and Raweta in Fig. 1, 2, and 4. The authors should try to perform the statistical analysis between different lines and discuss about that.
2) In Fig. 3, the authors showed SOD activity by native PAGE. How can you normalize the data? Please explain about it. Can the authors digitize the data? How about the data of oats and barley?
3) The reviewer would like to know symptoms of Mn excess in this condition. The authors should provide photographs in control and 10 mM MnCl2.
4) In Fig. 5, the authors provided microscopic data. It is difficult to see the structural changes in plastid and starch accumulation in this version. The authors should try to use TEM to show the data more clearly. How about the data of oats and barley?
Minor points
1) Are font size in some sentences bigger? P.1,L.16-18 and L.23-24, P.10,L.73-74, P.11, L.120-121 and L.127-128?
2) P.1, L.14 Write 'manganese (Mn)', then the authors can replace 'manganese' to 'Mn'. For example, P.3, L.102 and L.105, P.4, L.170, P.5,L.182, and others.
3) P.1, L.32 ), L.34 (. Delete parentheses.
4) P.3, L.110, P.4, L.150 Gravity unit should be written in italic.
5) Fig. 2 It is too small to see letters in graphs.
6) Author contributions are correct?
7) Grant information should be written in 'Funding'.
8) No acknowledgements? Then the section should be deleted.
