Responses of Cell Wall Components to Low Nitrogen in Rapeseed Roots
Round 1
Reviewer 1 Report
The authors have stidied the impact of low nitrogen supply on rapessed cell walls. The paper could bring new interesting information however, I have major concerns.
According to the protocol described by Hu and Brown, the extraction process leads to eight fractions : 1.5% SDS at 22°C for 2 h, 0.05 M CDTA (pH 6.5) at 22°C for 6 h; 0.05 M Na2CO3 at l°C ovemight and then 0.05 M Na2C03 at 2OoC for 3 h, 1 N KOH at 2O°C for 2 h under N2, 4 N KOH at 2O°C. The authors refer to this protocol : “the cell wall material was further separated into ion -bound pectin (ISP), covalently bound pectin (CSP), cellulose, hemicellulose and lignin as previously described by Hu and Brown”. Thereby, they obtained five fractions (against eight in Hu and Brown protocol) of which one contain lignins whereas Hu and Brown never refer to lignins… The way the authors have obtained the five fractions needs to be clarified and far more detailed. Otherway, it is impossible for reviewers and futire lectors to undertsand what authors have done.
line 164 “that low N treatment affected pectin monosaccharaides in rapeseed roots, with significant decrease of L-fructose, D-fructose, and D-galacturonic acid.”. Fructose is not a pectic monosaccharide and could come from the sucrose used for the cell wall extraction...
Figure 4: What about galactose and xylose monosaccharides? “Relative” pectin monosaccharide content : relative to what?
Last, I think that performing a TFA hydrolysis on Alcohol Insoluble Residue for each samples would have been necessary before performing all the sequential extraction process
Minor comment.
Thorough the manuscript: monosaccharide and not monosaccharaide, pectin lyase and not pectin layse
Author Response
The authors have studied the impact of low nitrogen supply on rapeseed cell walls. The paper could bring new interesting information however, I have major concerns.
Response: Thank you very much for your worthy suggestions and helpful comments. We have thoroughly revised the manuscript according to your suggestions and carefully answered your questions and comments point-by-point. We hope you will be satisfied with our revisions and responses.
- According to the protocol described by Hu and Brown, the extraction process leads to eight fractions: 1.5% SDS at 22°C for 2 h, 0.05 M CDTA (pH 6.5) at 22°C for 6 h; 0.05 M Na2CO3 at 1°C Ove might and then 0.05 M Na2CO3 at 20°C for 3 h, 1 N KOH at 20°C for 2 h under N2, 4 N KOH at 20°C. The authors refer to this protocol: “the cell wall material was further separated into ion -bound pectin (ISP), covalently bound pectin (CSP), cellulose, hemicellulose and lignin as previously described by Hu and Brown”. Thereby, they obtained five fractions (against eight in Hu and Brown protocol) of which one contain lignin whereas Hu and Brown never refer to lignin… The way the authors have obtained the five fractions needs to be clarified and far more detailed. Other way, it is impossible for reviewers and futire lectors to understand what authors have done.
Response: Thank you very much for pointing these questions. First, we are sorry for the wrong reference (Hu and brown) about the cell wall components extraction and it has been replaced by the right reference (Redgwell and Selvendran). In addition, we carefully checked the determine method of lignin and found lignin content was obtained according to the method described by Papa et al. (2012) and Pauly et al. (2008) instead of the method previously described in manuscript. Now it has been corrected in revised manuscript. To make the methods of cell wall components extraction and determination clearer and easier to understand for reviewers and readers, more details have been added to revision.
- line 164 “that low N treatment affected pectin monosaccharaides in rapeseed roots, with significant decrease of L-fructose, D-fructose, and D-galacturonic acid.”. Fructose is not a pectic monosaccharide and could come from the sucrose used for the cell wall extraction...
Response: You raised a very important question. Thanks a lot. As you said, fructose is not a pectin monosaccharide and could come from the sucrose used for the cell wall extraction. Considering that, our data of pectin monosaccharaides are problematic, thereby, we deleted this part and we will look for new methods to avoid the interference of some monosaccharides in future study.
- Figure 4: What about galactose and xylose monosaccharides? “Relative” pectin monosaccharide content: relative to what?
Response: In present study, a qualitative rather than quantitative method was used to determine pectin monosaccharide content, and the substances below the detection line cannot be detected. Thus, we did not get the content of galactose and xylose monosaccharide maybe resulted in their contents were below the detection line. And because the monosaccharide content was not quantitative, so we defined it as relative content. As we have responded in question 2, the results of pectin monosaccharides have been removed from revised manuscript.
- Last, I think that performing a TFA hydrolysis on Alcohol Insoluble Residue for each sample would have been necessary before performing all the sequential extraction process
Response: Thank you for your good advice. In future study, we will first to perform a TFA hydrolysis on Alcohol Insoluble Residue for each sample before performing all the sequential extraction process.
- Minor comment.
Thorough the manuscript: monosaccharide and not monosaccharaide, pectin lyase and not pectin layse
Response: The wrong points have been corrected according to your comments.
Reviewer 2 Report
The Ms (agronomy-1618531) entitled « Responses of cell wall components to low nitrogen in rapeseed roots » by Tian et al., reports that rapeseed plants, grown under low nitrogen content, increase root length and that is characterized by cell wall remodeling at a pectin and cellulose levels.
The paper might be interesting but some results must be clarified and experiments must be better described.
Tian et al should present the degree of methylation instead of demethylation since the experimental procedure used, is based on the measurement of the degree of methylation and not its demethylation.
Another issue is the monosaccharides content of the pectin. Did the authors show (Fig 4A) the profil of the ISP or CSP ?
Furthermore, there is no D- or neither L-Fructose in the cell wall.
That profile does not show all the pectic monosaccharides as rhamnose, galactose, glucuronic acid residues…that should be added.
Furthermore the authors showed β-galactosidase activity. That could arise from pectin but also from hemicellulose as xyloglucan or from AGP.
Did the authors measure pectin or pectate lyase since the kit used, is dedicated to pectate lyase activity ?
Page 9 : « had no remarkable effect on hemicellulose ». The authors should reformulate the sentence. The hemicellulose (HC) content does not likely change but the composition of HC may change as more/less galactose residues, for instance. That is the same comment for lignin content. The lignin composition may change.
The experimental procedure particularly the sequential extraction of the cell wall is waggy and that should be described.
Since the authors do not discuss the photosynthetic pigment results, they should therefore, move them to the supdata.
Minor comments :
Page 5, « 2.4 : monosaccharaide », please remove « a »
Page 4, please add a space between lignin and content
Page 2, the research materialto, please rephrase the sentence
Author Response
The Ms (agronomy-1618531) entitled « Responses of cell wall components to low nitrogen in rapeseed roots » by Tian et al., reports that rapeseed plants, grown under low nitrogen content, increase root length and that is characterized by cell wall remodeling at a pectin and cellulose levels.
The paper might be interesting but some results must be clarified and experiments must be better described.
Response: Thank you very much for your worthy suggestions and helpful comments. We have thoroughly revised the manuscript according to your suggestions and carefully answered your questions and comments point-by-point. We hope you will be satisfied with our revisions and responses.
- Tian et al should present the degree of methylation instead of demethylation since the experimental procedure used, is based on the measurement of the degree of methylation and not its demethylation.
Response: Pectin demethylation is beneficial to cell wall loosing, so comparing to the degree of pectin methylation, the degree of pectin demethylation is more intuitive to reflect the effect of low nitrogen on cell walls. Thus, we calculated pectin demethylation degree according to this formula: pectin demethylation degree=100- pectin methylation degree. Thank you and we hope you will be satisfied with our response.
- Another issue is the monosaccharides content of the pectin. Did the authors show (Fig 4A) the profile of the ISP or CSP?
Response: According to the comment of another reviewer on pectin monosaccharides:“fructose is not a pectin monosaccharide and could come from the sucrose used for the cell wall extraction.”. So we think maybe the results were problematic, we have deleted this part from revised manuscript and we will look for new methods to avoid the interference of some monosaccharides in future study.
- Furthermore, there is no D- or neither L-Fructose in the cell wall.
That profile does not show all the pectic monosaccharides as rhamnose, galactose, glucuronic acid residues…that should be added.
Response: thank you for pointing out this question. In present study, a qualitative rather than quantitative method was used to determine pectin monosaccharide content, and the substances below the detection line cannot be detected. Thus, we did not get the content of rhamnose, galactose, glucuronic acid residues maybe resulted in their contents were below the detection line. And because the monosaccharide content was not quantitative, so we defined it as relative content. We will look for new methods to avoid the interference of some monosaccharides in future study to make the results more scientific and reliable.
- Furthermore the authors showed β-galactosidase activity. That could arise from pectin but also from hemicellulose as xyloglucan or from AGP.
Response: Changes on β-galactosidase activity could arise from pectin but also from hemicellulose. In present study, low N had no obvious effect on hemicellulose content, so we just considered the effect of β-galactosidases on cell wall pectin degradation. We also read many published articles about β-galactosidase and pectin-degrading function (Esteban et al., 2003, Plant Cell Physiology; Gwanpua et al., 2014, Food Chemistry; Huang et al., 2026, Carbohydrate Polymers).
- Did the authors measure pectin or pectate lyase since the kit used, is dedicated to pectate lyase activity?
Response: We have checked it and confirmed that it should be pectate lyase instead of pectin lyase. The mistake also has been corrected in the corresponding position of the revised manuscript.
- Page 9: « had no remarkable effect on hemicellulose ». The authors should reformulate the sentence. The hemicellulose (HC) content does not likely change but the composition of HC may change as more/less galactose residues, for instance. That is the same comment for lignin content. The lignin composition may change.
Response: Thank you. The sentence has been revised ad bellow: “Our results also showed that low N stress changed the cell wall composition of rapeseed roots, accompanied by a significant increase in two different type pectin and cellulose content, but had no remarkable effect on hemicellulose and lignin content (Fig. 3C, E-G)”.
- The experimental procedure particularly the sequential extraction of the cell wall is waggy and that should be described.
Response: To make it clearer and easier to understand for reviewers and readers, more details on the methods of cell wall components extraction and determination have been added to revised manuscript.
- Since the authors do not discuss the photosynthetic pigment results, they should therefore, move them to the supdata.
Response: We have discussed the photosynthetic pigment results in “Discussion” section. Thank you.
- Minor comments :
Page 5, « 2.4 : monosaccharaide », please remove « a »
Page 4, please add a space between lignin and content
Page 2, the research materialto, please rephrase the sentence
Response: according to your good suggestions, we have removed «a» from «monosaccharaide»，added a space between lignin and content and rephrase the sentence in page 2.
Round 2
Reviewer 1 Report
The authors have reply to my previous comments. However, it would have been quite better to perform as requested a TFA hydrolysis followed by a monosaccharide composition to make the manuscript more substantial.
