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［题外话］最近读了很多博主的文章，深有感触，所以将我以前没有任何价值，甚至是污染人们眼睛的博文统统删除了。现在我想发一些我认为有价值的博文，尽量让大家觉得看我的博文不是在浪费时间。
我是个论文新手，请不要笑话我！
Dear editor and reviewers,
We really appreciate your earnest and careful review of my manuscript entitled as "XXXXXXXXX".
The language was polished as required and the constructive comments from both reviewers were appreciated and carefully considered with a complete revision. Please check the revised manuscript and responses below for more details.
Again, many thanks for your efforts making our manuscript much better!
Best regards.
Guo-Ming Shen, Ph.D.,
School of Food Science and Biotechnology
Zhejiang Gongshang University
149 Jiaogong Road, Hangzhou, 310012
China.
Reviewer: 1
Recommendation: Accept paper with major/complete revision
Comments:Review result of MS jph.201000310 by Shen et al.This study was to examine the responses of a high Cd accumulating rice mutant to Cd exposure, including Cd contents, antioxidants, antioxidative enzyme activity, in roots. A mutant has been successfully obtained cadH-5. Although the physiological and biochemical investigations have been done in rice upon exposure to Cd, it is still interesting for using a high Cd accumulating rice mutant to study of Cd responses.Because authors did not cite references mentioning the responses of shoots of rice seedlings to Cd administration in root system, the readers do not have any idea for why only roots were assayed. Authors have to explain this in the Introduction. Another concern is that the backgrounds on the relationships between Cd and rice, and between Cd and oxidative stress in rice. It is not easy for readers to understand why this investigation is necessary. Several experimental methods have not been carefully described. It can not be accepted in the present form. The data are not well calculated and presented in Figures. For example, in the Figure 2 and Figure 4 b, the Y-axis was drawn from a value < 0? I strongly suggested that other Figures are also revised. In addition, because bar was chosen for Figure 3 and Figure 4a, the data of Y-axis should be from 0. It is a mistake.It needs a major revision. All comments should be considered before it can be accepted for publication in the Journal.
Response: We have carefully revised the manuscript, with all problems and concerns mentioned above have been considered and solved (see the revised manuscript for details), such as the relationships between Cd and rice, and between Cd and oxidative stress in rice, and all figures. Below we listed our responses to each comment one by one. Abstract1. P. 2, line 12-13: ‘But, Cd accumulation in roots organelles varied.’ needs to be clarified. It is not adequate for the use of ‘accumulation’ here.
Response: We should have used the 'contents' instead of 'accumulation'. We corrected all misuses of ‘accumulation’ in the text.
2. P. 2, line 16: The full name of GSH/GSSG and ASC/DHA is needed in the Abstract, although these have been mentioned in the Introduction and Materials and Methods?Response: The full names were added as your request.
Introduction1. The Introduction is needed to be re-written, especially the backgrounds on the relationships between Cd and rice, and between Cd and oxidative stress in rice. It is not easy for readers to understand why this investigation is necessary.
Response: We have re-written this section and highlighted the changes with track.
2. P. 3, line 9: changed to ‘Cd can also affect different metabolic processes, ‘
Response: Corrected.
Materials and Methods1. It needs to be carefully revised. Many usages and sentences are not adequate. Some is listed as follows.2. P. 4, line 12: japonica -> Japonica
3. P. 4, line 23: (photo flux density of 500 μM•s-1•m-2). -> (photon flux density of 500 μmol•m-2•s-1). The light source should be also indicated.
4. P. 5, lines 8-9: with the wild type Kasa (Oryza sativa ssp. Indica -> with the wild type Indica rice variety Kasa
5. P. 5, line 15: Hydroponic Culture …: Authors stated that 0 d as the control. According to the design and results of the present study, the data of 0 d were the initial value as the control, that is, the initial control, and for example, WT is the control material in Figure 2. So, ‘0 d (as control)’ is not adequate. Authors have to correct it.
6. P. 5, line 17: 0 Mm (as control) -> 0 mM (as control)
P. 5, line 20: For, ‘the roots were cut, frozen in liquid nitrogen’, some free Cd2+ may be carried on the surface of root tissue. In general, to assay the Cd contents in tissues treated with Cd2+, tissues will be washed with Milli-Q water to remove the surface Cd2+. Authors should clarify whether the roots are rinsed with Milli-Q water or not.
8. P. 5, line 23: Because the uptake of Cd was not really calculated in the present study, so changed Cd uptake -> Cd contents
P. 6, line 2: were measured AAS directly -> were measured by AAS directly
10. P. 6, line 25: For H2O2 determination, the unit was μmol?g-1 of dry weight (DW). Authors did not show how the root dry weight was determined or estimated? It has to be indicated.
11. P. 8, Enzyme Assay: changes ‘absorption coefficient’ -> ‘extinction coefficient’ in this section
P. 8, line 11: changes to ‘potassium phosphate buffer (pH 7.8) containing …’
P. 8, line 15: changed to ‘Aliquot of the supernatant (50 ?L) was ….’ (also in line 24)
P. 9 Statistical Analysis: No statistical method has been indicated. It seems that only t-test has been approached in this study. If yes, it has to be indicated here or in Figure legend.Response: All the typos and grammars (from 1-14) were corrected as suggested.
Results and Discussion1. Changed H-5 -> cadH-5 in Figure 1.
P. 9, line 17: 0.75 mM Cd -> Cd of 0.76 mM
Response: They have been corrected as suggested.
P. 9, lines 17-20: Why authors stated it is reasonable for the choice of 83.25 g Cd•g-1 as screen level. They said tat this is based on the results of (Bingham et al., 1975) that rice was still alive on the concentration of 640 g Cd/g soil. Because these are two different units, it is hard for readers to make sense. It has to be clarified and re-written.
Response: We completely agree with the reviewer. It is unnecessary statement, and we have deleted this sentence.
4. P. 10, line 5: mM? Check the concentration unit is the Italic font?
P. 10, line 7: statistical significant, indicated -> statistical significance, indicating
P. 10, line 8: changed to ‘ a relatively high Cd accumulation mutant’
P. 10, Figure 2: the Y-axis is drawn from a value < 0? It is a big mistake. Authors have to check their data carefully.
P. 10, line 9: Cd-PC: In general, PC was calculated from non-protein thiol. But authors did not indicate this in Materials and Methods. (move P. 11, lines 27-28 to Materials and Mehods)
P. 10, lines 8-13: Because the shoots or straws or leaves were not the materials for the assay of Cd contents, it is not known whether Cd contents will be relatively high in shoots in mutant as compared to WT shoots. I am wondering that can authors state that the straw is often fed to ruminants; thus, the mutant cadH-5 is a potential hazardous material? It seems to me that authors have over-explained or over-extended their results. By the way, because authors would like to explain the responses of rice, the cited references (lines 10-11) should be restricted to rice instead of other plants.
10. P. 10, line 14: Specify the tissue as root: The subcellular distribution of Cd showed -> The subcellular distribution of Cd in roots showed.
P. 10, line 15: The increase in Cd -> The increase in Cd contents
12. P. 11, lines 4-26: Several references on H2O2 and Cd toxicity in rice have not been cited and discussed. For example, Shah K, Kumar RG, Verma S, et al. 2001. PLANT SCIENCE 161: 1135-1144; Kuo MC, Kao CH. 2004. BOTANICAL BULLETIN OF ACADEMIA SINICA (presently Botanical Studies) 45: 291-299; Hsu YT, Kao CH. 2007. PLANT AND SOIL 298: 231-241, 300: 137-147; Chao YY, Hsu YT, Kao CH. 2009. PLANT AND SOIL 318: 37-45
13. P. 11, lines 27-30 and P. 12, lines 1-3: PC has been well known in heavy metal detoxification. Authors did not cite references and discuss here, therefore it is strongly recommended authors to compare their results with published data in references, even in plants other than rice.（呵呵！审稿人估计就是这个人，不过不引用他的文章是我的不对，是我忽视了他的工作。）
14. P. 12, line 2: Revise ‘For, Cd can combine phytochelatins’, and it should be ‘phytochelatins can bind Cd’
15. The data of Y-axis in Figure 3 should be from 0, because it is ‘bar’.Reference:P. 19, line 14: Italic font for Brassica juncea
P. 19, lines 17-19: Capitolize the first character of each word?
Response: Thanks for the reviewers careful insights! All the comments and suggestions (4-15 from Results and Discussion, References) were carefully considered and revised accordingly.
Reviewer: 2
Recommendation: Accept paper with major/complete revision
Comments:Environmental and Experimental BotanyManuscript Number: EEB-D-09-00400 (We were a little confused by the Manuscript Numberhere (EEB-D-09-00400). I thought My MS Number is jph.201000310.（晕死了，审稿人将我们文章投的刊物搞错了）)Title: XXXXXXXXXXXXX
Guo-Ming Shen, et al. The paper bases on the fact that an environmental stress such as heavy metal stress, NaCl salinity nutrient deficiency can lead to an increase of cellular damage due to increased ROS generation. The relationship between stress, antioxidative activity and tolerance was lately reported in several crops and has become a major topic for research. However, studies related to Oryza sativa antioxidative system towards Cd stress conditions are scare. The authors showed impressively by comparing a wiltype (WT) with a mutant (cadH-5) that oxidative stress plays an important role at Cd detoxification and that under these conditions several antioxidative mechanisms act together in a distinct pattern.Beside the interesting story of this paper I have also some criticism according to this basic aspect. The selected parameters do not allow a clear causal explanation for differences in tolerances between wildtype and mutant! Heavy metal stress is a multifactorial burden for the plants and the ions play the (!) major role if you want to distinguish between Ca or Mg deficiency and Cd toxicity. I would emphasize to add results about ion relations to optimize the discussion.
Response: Thanks for your insightful suggestions! However, heavy metal stress is a complex biological process, we can not present ion relation results in this paper due to space limit. Recently, Liu et al. (2003a, 2003b see ref.) demonstrated that neither sensitive nor tolerant rice cultivars/genotypes had significant correlation between Cd and Mg. Furthermore, in this experiment all element is excessive in rice culture solution, we believe that Cd can not cause Ca or Mg deficiency in rice. Using GeoChem-EZ software (Shaff et al., 2010. Plant Soil 330, 207-214) results as following:
Ca ( 87.54 % as a free metal; 0.16 % complexed with PO4; 0.73 % complexed with Citr; 0.01 % complexed with Cl; 11.57 % complexed with SO4)
Mn +2
86.99 % as a free metal
1.03 % complexed with Citr
.48 % complexed with Cl
11.49 % complexed with SO4
Cd
56.17 % as a free metal
.04 % complexed with PO4
23.36 % in solid form with PO4
.71 % complexed with Citr
7.91 % complexed with Cl
.06 % complexed with NO3
11.76 % complexed with SO4
Zn
73.88 % as a free metal
.25 % complexed with PO4
15.61 % complexed with Citr
.26 % complexed with Cl
.09 % complexed with NO3
9.76 % complexed with SO4
.14 % complexed with OH-
Mg
89.85 % as a free metal
.03 % complexed with PO4
.57 % complexed with Citr
.12 % complexed with Cl
9.43 % complexed with SO4
K
99.19 % as a free metal
.03 % complexed with Cl
.77 % complexed with SO4
Additionally are the described antioxidative responses a consequence of problems with or at least connected to the CO2/H2O gas exchange. I am astonished that the authors did not consider this at all! I would expect to find this aspect much more pronounced in the introduction and in the discussion. I would also emphasize to include results about CO2/H2O gas exchange (photosynthesis) or at least about chlorophyll fluorescence. This aspect is conspicuously missing apart from that impressing list of results.
Response: We should have added something we already knew about CO2/H2O gas exchange in this manuscript. Former experiments in our Lab demonstrated (He et al. 2008, Photosynthetica 46, 466-470) that in the sensitive rice mutant (now named as cadB-1) had lower net photosynthetic rate (PN), transpiration rate (E), and stomatal conductance (gs) than WT rice, however, it had higher intercellular CO2 concentration (Ci), indicating that non-stomatal factors accounted for the inhibition of PN. Maximal photochemical efficiency of photosystem 2 (Fv/Fm), effective quantum yield of PS2 (ΦPS2), and photochemical quenching (qP) decreased much in the mutant under Cd stress.
There is another similarity between introduction and discussion which is really annoying: The style of writing is extremely static and there are several grammar mistakes! But let me discuss the content of both chapters:1) The uptake by divalent ion transporters is just mentioned but its consequence not considered at all.
Response: We deleted the sentence "Cd is taken up by divalent ion transporters in the roots." in the chapter of Introduction. And we will further discuss this in our next manuscript as "Interaction of Cd and mineral nutrients for uptake and accumulation in rice Cd sensitive cadB-1 and Cd tolerant cadH-5 mutants."
2) The applied aspect or better the intension of this work is not explained sufficiently. If you produce a more tolerant but at the same time more Cd- accumulating rice mutant you also develop a more toxic food. Wouldn´t it make more sense to select and develop an avoidance type?
Response: In the revised MS, we presented it as "The mutant might be a good material for phytoremediation in heavy metal contamination rice planted area."
3) Your major argument for ROS stress is that Cd “can indirectly activate the plasma–membrane reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and generate reactive oxygen species”. However, the results of NADP, NADPH (or the ratios of) do not fit perfectly to your results of the enzymes and substrates involved in the Halliwell-Asada system (see also page 12 line 21 to 25, page 14 line 23 to 30)?!
Response: We agree with the reviewer and have re-written this section. Please check it for more details in the text.
4) In addition to this Achilles' heel you argue that ASC-GSH cyle was more inhibited in the WT than in the cadH-5 mutant. I would like to know how you can distinguish between a higher expression in the mutant and an inhibition in the wildtype?
This static system is visible in all chapters, in the material and methods such as in the discussion. It is common to use such a static system in the chapter results, so it is there not as annoying as in the other chapters! In the discussion it is nothing else but a list of a short interpretation of every single result – one after the other in the same sequence as in the chapter results. Partially important bits of the discussion are missing: This is also the case e.g. for the interpretation of the differences between ROS and NADPH development (page 14 line 5 to 10). Please add this discussion and in this aspect also some essential literature!The single discussions are incomplete, they do not give a survey of the relevant corresponding literature and they do not give a complete survey about potential relationships and the conception of causal relation. Therefore the overall message remains far behind the potential of the shown results!!
I have also my problems to understand the direct connection to the interrogation and importance of the subcellular distribution analysis of Cd. If you include it to the text, it should be discussed in a comprehensible manner. This is not done at all. The reason is probably a missing link in the argumentation chain. This could be the connection to other divalent ions!
Response: Appreciate for the comments. We realized this and have re-written the section of Results and Discussion.
I am completely happy about the used statistics with one exception. I cannot comprehend the number of replicates used! Please add this information (number of cultures etc.).
Response: The numbers of replicates used were added in the text.
At the end just some formal aspect: The authors should control critical the grammar of their text (such as at page 2 line 12 or page 3 line 20 and 23. In general, the paper is clear and well-written although it would help to ask a native English speaker to improve the grammar! However, because of the reasons mentioned above it is suitable for publication just after major corrections.
Response: Thanks for your suggestions. We got some native English speakers to improve the language.
看到没有，这两个审稿人多认真，是他们在指导我写论文。国内的所谓博导多半不会指导学生这样写文章吧！
所以博士生们，你们的导师如不参与写作、修改论文，我建议将文章投向德国刊物，看他们多认真！
https://blog.sciencenet.cn/blog-4528-394118.html
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