Genome-Wide Analysis of the β-Amylase Gene Family in Brassica L. Crops and Expression Profiles of BnaBAM Genes in Response to Abiotic Stresses

Round 1

Reviewer 1 Report

This paper presents an analysis of the beta amylase gene family in three related species of Brassica.  All of the methods used are conventional and straightforward.  Most of my comments concern English grammar or presentation of the figures. The font in all of the figures is far too small for print versions of these figures. 

The only scientific issue I have is on lines 343-345, where the authors suggest that because there is one fewer BAM genes in B. napus than in both parent species, the loss must have occurred during a whole genome duplication in the parents.  I do not understand this argument. If the gene was lost in one of the parents, the number of BAM genes in B. napus would not be affected.  It seems more likey that the loss could have occurred at anytime in the B. napus lineage.

Minor comments - 

line 14 - insert 'and' between ... (B. rapa), and 20 BoBam genes ...

line 18 - insert 'and between ... collinearity and the sequence alignment ...

line 36 contains a run-on sentence.  It should read ...stress response.  For instance, the starch content ...

line 41 should read  ...soluble sugars [2,6,17,18], suggesting that ...

line 53 another run-on sentence.  Should read ...growth and development. These unique features ...

line 57 - 59  Shouldn't these genes be StBam  and not SrBam?

line 86 - 'allotetraploids' should be 'allotetraploid'

line 90 - Citation needed for B. napus genome

lines 131 and 133 - 'camber' should be 'chamber'

line 147 - '... study were listed ...'  should be '... study are listed ...'

line 163  insert a space after mean _ +/-SD

line 169 - run on sentence. Should read ... database. A total of 30 BAM ...

line 174  'had less homologs' should be 'had fewer homologs'

line 176 insert 'and.'  ... amino acids, and the protein molecular ...

Table 1 - What does 'random' mean when its is used as part of the chromosomal location?

Figure 1 - The font is far too small for a printed version of this figure. The legend should indicate that genes from different species are color coded within each family.

Figure 2 - The font is far too small for a printed version of this figure.

Figure 3 - The font is far too small for a printed version of this figure.

Figure 4 - The font is far too small for a printed version of this figure.

line 262 Spell out the word 'Eight' to begin the sentence.  Also on this line, the word 'less' should be replaced with 'fewer.' ( This difference frequently causes problems for native English speakers.)

Figure 5, line  267 - I don't understand when the authors say that genes with a syntenic relationship are linked with lines in different color.  Do you mean that groups of genes with syntenic relationships are linked with lines of the same color?

Line 286 -  ... widely known for its involvement ... should read widely known for their involvement ...

Figure 6 - Font is waaaaay to small.

Line 318  'severer'  should be 'severe'

Figure 7 - Font is too small

Line 369 - 'progresses'  should be 'progress'

 

 

 

Author Response

This paper presents an analysis of the beta amylase gene family in three related species of Brassica.  All of the methods used are conventional and straightforward.  Most of my comments concern English grammar or presentation of the figures. The font in all of the figures is far too small for print versions of these figures. 

R: Thank you for the valuable suggestions, the English writing of this manuscript has been improved which are highlighted in red. The front of all figures have been revised.

The only scientific issue I have is on lines 343-345, where the authors suggest that because there is one fewer BAM genes in B. napus than in both parent species, the loss must have occurred during a whole genome duplication in the parents.  I do not understand this argument. If the gene was lost in one of the parents, the number of BAM genes in B. napus would not be affected.  It seems more likey that the loss could have occurred at anytime in the B. napus lineage.

R: Thank you for the comments. We have corrected the description in line 366.

Minor comments - 

line 14 - insert 'and' between ... (B. rapa), and 20 BoBam genes ...

R: Thank you for the suggestion. We have revised, line 14.

line 18 - insert 'and between ... collinearity and the sequence alignment ...

R: We have revised, line 18.

line 36 contains a run-on sentence.  It should read ...stress response.  For instance, the starch content ...

R: We have corrected the mistake, line 38.

line 41 should read  ...soluble sugars [2,6,17,18], suggesting that ...

R: We have revised, line 44.

line 53 another run-on sentence.  Should read ...growth and development. These unique features ...

R: We have revised the sentence, line 55.

line 57 - 59  Shouldn't these genes be StBam  and not SrBam?

R: Yes, it should be StBam. We have corrected the mistakes, line 59.

line 86 - 'allotetraploids' should be 'allotetraploid'

R: We have corrected the mistake, line 90.

line 90 - Citation needed for B. napus genome

R: We have added the citation, line 90.

lines 131 and 133 - 'camber' should be 'chamber'

R: We have corrected the mistakes, line 137.

line 147 - '... study were listed ...'  should be '... study are listed ...'

R: We have corrected the mistakes, line 155.

line 163  insert a space after mean _ +/-SD

R: We have corrected the mistakes, line 171.

line 169 - run on sentence. Should read ... database. A total of 30 BAM ...

R: We have revised the sentence, line 176.

line 174  'had less homologs' should be 'had fewer homologs'

R: We have corrected the mistakes, line 181.

line 176 insert 'and.'  ... amino acids, and the protein molecular ...

R: We have revised, line 183.

Table 1 - What does 'random' mean when its is used as part of the chromosomal location?

R: The genome-related data of B. napus (Darmor-bzh) were derived from http://www.genoscope.cns.fr, ‘random’ means that the gene was not assembled into the specific location of the genome, thus the position after ‘random’ only has certain significant reference. We have clarified the description in method (line 110).

Figure 1 - The font is far too small for a printed version of this figure. The legend should indicate that genes from different species are color coded within each family. Figure 2 - The font is far too small for a printed version of this figure. Figure 3 - The font is far too small for a printed version of this figure. Figure 4 - The font is far too small for a printed version of this figure.

R: Thank you for the valuable suggestions, we have improved the figures and figure legend.

line 262 Spell out the word 'Eight' to begin the sentence.  Also on this line, the word 'less' should be replaced with 'fewer.' ( This difference frequently causes problems for native English speakers.)

R: We have corrected the mistakes, line 270.

Figure 5, line  267 - I don't understand when the authors say that genes with a syntenic relationship are linked with lines in different color.  Do you mean that groups of genes with syntenic relationships are linked with lines of the same color?

R: Yes, the figure legend means groups of genes with syntenic relationships are linked with lines of the same color. We have corrected the mistake in line 276.

Line 286 -  ... widely known for its involvement ... should read widely known for their involvement ...

R: We have corrected the mistake, line 296.

Figure 6 - Font is waaaaay to small. Figure 7 - Font is too small

R: we have improved the figure 6 and figure 7.

Line 318  'severer'  should be 'severe'

R: We have corrected the mistake, line 334.

Line 369 - 'progresses'  should be 'progress'

R: We have corrected the mistakes, line 392.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript presents the characteristics of the BAM gene family in Brassica napus, B. oleracea and B. rapa. The first thing I would like to suggest is to correct the title because as it stands it suggests that the subject of the article was only B. napus.

In the manuscript authors indicated that 30 BAM-family genes in B. napus, 11 in B. rapa, and 20 in B. oleracea were identified, however in further analysis of BAM proteins sequences in B. napus it was clearly shown, that not all of the potential proteins will have catalytic activity, therefore some of the genes are encoding so called beta-amylase-like proteins rather than beta-amylase. I think that the fact should better accented in presented work.

Moreover , some authors suggest that on the basis of introns placement BAM gene family  should be divided into 2 sub-families rather than four as was previously thought (Monroe JD, Breault JS, Pope LE, Torres CE, Gebrejesus TB, Berndsen CE, et al. Arabidopsis β-amylase2 is a K+-requiring, catalytic tetramer with sigmoidal kinetics. Plant Physiol. 2017;175:1525–35.; Monroe JD, Storm AR. The Arabidopsis β-amylase (BAM) gene family: diversity of form and function. Plant Sci. 2018;276:163–70.). I think, that even if authors disagree with such statement, it should be mentioned and addressed in the discussion.

I also have a few questions about materials and treatments section. How were the plants grown?  On which medium? In pots? What was the intensity of light? Moreover, I think that it would be beneficial if the intensity of stress was also measured by some parameters (in example RWC for drought), because now it is not clear if after 1h of treatment the plants were indeed stressed. How were such parameters as photoperiod and temperature assured in greenhouse?

I also have a few comments regarding the presentation of the results. In Figure 1, the names of genes against a green background are completely unreadable even at high magnification. I suggest changing the color in such a way to make the drawing clearer. I have similar comments on Figure 6. The font in Figure 6 is so small that it is completely unreadable.

My doubts are also raised by the interpretation of the results regarding the starch content and beta-amylase activity. In lines 314-316 the authors argue that drought, flood, heat and cold treatment decrease the starch content, while the graphs below (Fig. 7) clearly show that e.g. when heat applied, the starch content initially increased, then decreased, and then returned to its original level. In line 320-321 the authors state that stress stimulates the activity of beta-amylases, while in Figure 8 the activity of these enzymes during stress treatment is the same or decreasing in comparison to the level before its application.

Author Response

The manuscript presents the characteristics of the BAM gene family in Brassica napus, B. oleracea and B. rapa. The first thing I would like to suggest is to correct the title because as it stands it suggests that the subject of the article was only B. napus.

R: Thank you for the valuable suggestion, we have changed the title to “Genome-wide analysis of the β-amylase gene family in Brassica crops and expression profiles of BnaBAM genes in response to abiotic stresses”.

In the manuscript authors indicated that 30 BAM-family genes in B. napus, 11 in B. rapa, and 20 in B. oleracea were identified, however in further analysis of BAM proteins sequences in B. napus it was clearly shown, that not all of the potential proteins will have catalytic activity, therefore some of the genes are encoding so called beta-amylase-like proteins rather than beta-amylase. I think that the fact should better accented in presented work.

R: Thank you for the comments, we have clarified the description in the manuscript (line 19, 225).

Moreover , some authors suggest that on the basis of introns placement BAM gene family  should be divided into 2 sub-families rather than four as was previously thought (Monroe JD, Breault JS, Pope LE, Torres CE, Gebrejesus TB, Berndsen CE, et al. Arabidopsis β-amylase2 is a K+-requiring, catalytic tetramer with sigmoidal kinetics. Plant Physiol. 2017;175:1525–35.; Monroe JD, Storm AR. The Arabidopsis β-amylase (BAM) gene family: diversity of form and function. Plant Sci. 2018;276:163–70.). I think, that even if authors disagree with such statement, it should be mentioned and addressed in the discussion.

R: Thank you for the valuable suggestions, these two recent studies performed evolutionary analysis based on shared intron positions and sequence alignments, and the present manuscript carried sequence alignments based on amino acid. Therefore, the number of subfamily was different. Now we have clarified the description in the discussion part (line 360-363).

I also have a few questions about materials and treatments section. How were the plants grown?  On which medium? In pots? What was the intensity of light? Moreover, I think that it would be beneficial if the intensity of stress was also measured by some parameters (in example RWC for drought), because now it is not clear if after 1h of treatment the plants were indeed stressed. How were such parameters as photoperiod and temperature assured in greenhouse?

R: Thank you for the comments. We have revised the materials and treatments section, which included the growth process and condition. Actually, both the long-term cold stress and short-term cold stress were performed in chamber, we are sorry for the incorrect using for greenhouse and phytotron, and have clarified the description (line 136-146). The sampling time during abiotic stress in this manuscript was according to previous studies [11, 19, 33, 39], which revealed that abiotic stresses indeed affect plant in a short time, these references have been cited in the method part. In addition, the current study aims to investigate how abiotic stresses affect starch metabolism and beta-amylase activity, therefore, we did not measure other traits such as the drought-related RWC.

I also have a few comments regarding the presentation of the results. In Figure 1, the names of genes against a green background are completely unreadable even at high magnification. I suggest changing the color in such a way to make the drawing clearer. I have similar comments on Figure 6. The font in Figure 6 is so small that it is completely unreadable.

R: Thank you for the suggestions, we have improved the figures according to your comments.

My doubts are also raised by the interpretation of the results regarding the starch content and beta-amylase activity. In lines 314-316 the authors argue that drought, flood, heat and cold treatment decrease the starch content, while the graphs below (Fig. 7) clearly show that e.g. when heat applied, the starch content initially increased, then decreased, and then returned to its original level. In line 320-321 the authors state that stress stimulates the activity of beta-amylases, while in Figure 8 the activity of these enzymes during stress treatment is the same or decreasing in comparison to the level before its application.

R: sorry for the confusing description, we have rewritten the result part (line 325-339). There was no direct association between starch accumulation and β-amylase activity, and we have provide the explanation in the discussion part (line 411-418).

Author Response File: Author Response.pdf