Transcriptome Analysis of a Multiple-Branches Mutant Terminal Buds in Betula platyphylla × B. pendula
Round 1
Reviewer 1 Report
The Manuscript by Han et al. is devoted to importamt problem: investigation of genes mechanisms of development of multiple-branches in birches and connection of these multiple-branches with physiological processes (photosynthesis, transpiration, growth, and others). Results of work are interesting; however, there are several minor questions and remarks:
1. P. 2, line 89: OE2 should be described in more details. Why this “transgenic control” used? How it is differed from wild type?
2. P. 3, line 108: Maybe: Pw = Dry weight / Fresh weight * 100%? “%” were used in Figure 1a.
3. P. 3, line 110: Stereomicroscope by Olympus should be specified (series, country of manufacturer, etc).
4. P. 3, lines 116-121: Measurements of photosynthesis and transpiration by Li-co-6400 should be described in details. In particular, were CO2 concentration, H2O concentration and temperature controlled? If yes then what values of these parameters were used? Was sunlight or artificial light used? If the artificial light was used then was spectral maximum and light intensity of the light? If sunlight was used then what was method of its measurement?
5. P. 5, line 181, P. 5, line 197, P. 5, line 211, etc: Why br was analyzed in comparison with averaged OE2 and WT? OE2 and WT can be significantly differed (e.g. Table 1). Was application of averaged OE2 and WT correct? It should be clarified.
6. P. 5, Figure 1: Was caption of y-axis (“Frequency, %) correct? I suppose that the y-axis in Figure 1a shows “Dry to wet ratio” and the y-axis in Figure 1b shows “Water content”.
7. P. 5, lines 211-213; Figures 2c, 3; Table S2: Ii is not clear: why stomata diameters and density were low in br, but transpiration and assimilation were increased in br? It should be clarified.
8. P. 5, line 214: Figures 3a, b, c, d do not show WUE.
9. P. 5, lines 211-213; P. 15-16, lines 404-406, Figures 2c, 12a.b: Why expression of photosynthetic genes was reduced in the br, but photosynthetic assimilation of CO2 was increased in the mutant? It should be clarified.
Thus, I suppose that minor revision is necessary.
Author Response
Response to Reviewer 1 Comments
Forests
April 12, 2019
Dear Reviewer，
Re: Manuscript reference No.forests-485631
Please find attached a revised version of our manuscript "Transcriptome Analysis of a Multiple-Branches Mutant Terminal Buds in Betula platyphylla × B. pendula".
Thank you very much for your comments and suggestions.We have revised the manuscript, according to the comments and suggestions of reviewers and editor, and point by point to, the comments as listed below. Revisions in the text are shown using revision mode for additions and deletions.
We have realized that“stomata diameters and density were low in br, but transpiration and assimilation were increased in br” and “expression of photosynthetic genes was reduced in the br, but photosynthetic assimilation of CO2 was increased in the mutant”as you mentioned were contradictory because the materials used to analyze the photosynthetic parameters, stomatal parameters and DEGs related to photosynthesis were in different years. Therefore, we had deleted the results about photosynthetic parameters and DEGs related to photosynthesis. However, other results could be used to identify br, including height, branches number and size of apical buds.
We hope that the revisions in the manuscript and our accompanying responses will be sufficient to make our manuscript suitable for publication in Forests.
Looking forward to hearing from you soon.
With kindest regards,
Yours Sincerely
The Manuscript by Han et al. is devoted to importamt problem: investigation of genes mechanisms of development of multiple-branches in birches and connection of these multiple-branches with physiological processes (photosynthesis, transpiration, growth, and others). Results of work are interesting; however, there are several minor questions and remarks:
1. P. 2, line 89: OE2 should be described in more details. Why this “transgenic control” used? How it is differed from wild type?
Response: Thanks for your comments. OE2 was selected from 19 over-expressing BpCCR1 lines. It could prove that the variations of br was not caused by the function of BpCCR1. We have clarified WT, OE2 and br in plant materials as follows:
A birch (Betula platyphylla × B. pendula) multiple-branches mutant br, BpCCR1 overexpression line OE2 and wild type birch (WT) were used as plant materials. All the plants were grown in the pots with dimensions of 35 cm × 35 cm under natural conditions at the breeding base of Northeast Forestry University, Harbin, China. br was a transgenic line of BpCCR1 overexpression birch that exhibited quite different phenotypes with other BpCCR1 transgenic lines, and OE2 was used for excluding the function of BpCCR1.
2. P. 3, line 108: Maybe: Pw = Dry weight / Fresh weight * 100%? “%” were used in Figure 1a.
Response: Thanks for your comments. We have changed “Pw = Dry weight / Fresh weight” into “Pw = Dry weight / Fresh weight×100% “ in line 124.
3. P. 3, line 110: Stereomicroscope by Olympus should be specified (series, country of manufacturer, etc).
Response: Thanks for your comments. We have added series and country of Olympus as follows: stereomicroscope SZX7 (Olympus, Tokyo, Japan) in line 126.
4. P. 3, lines 116-121: Measurements of photosynthesis and transpiration by Li-co-6400 should be described in details. In particular, were CO2 concentration, H2O concentration and temperature controlled? If yes then what values of these parameters were used? Was sunlight or artificial light used? If the artificial light was used then was spectral maximum and light intensity of the light? If sunlight was used then what was method of its measurement?
Response: Thanks for your comments. CO2 concentration was about 700 µmol· mol-1, relative air humidity was about 80%, and air temperature was about 22℃ at 6:00 am. The experiment was performed under sunlight, and the intensity was recorded under 6400-08 transparent base chamber. We felt sorry about not measuring the diurnal variation of CO2 concentration, relative air humidity and air temperature. However, we had deleted all parts about measurement of photosynthetic parameters, including method, result and discussion. The reasons were based on the response of Point 7 and Point 9.
5. P. 5, line 181, P. 5, line 197, P. 5, line 211, etc: Why br was analyzed in comparison with averaged OE2 and WT? OE2 and WT can be significantly differed (e.g. Table 1). Was application of averaged OE2 and WT correct? It should be clarified.
Response: Thanks for your comments. We have analyzed in comparison with OE2 and WT, respectively, not the average of WT and OE2 in line 220, line 221, line248, line249 line250 and line251. For example, we have correct “The leaf size and morphology of br changed obviously, which showed that the leaf area was decreased to 41.51% of the average of other two lines” to “The leaf area was decreased by 56.12% and 60.62% of WT and OE2, respectively.” in the revised manuscript.
6. P. 5, Figure 1: Was caption of y-axis (“Frequency, %) correct? I suppose that the y-axis in Figure 1a shows “Dry to wet ratio” and the y-axis in Figure 1b shows “Water content”.
Response: Thanks for your comments. We have changed y-axis in Figure 1a, 1b.
7. P. 5, lines 211-213; Figures 2c, 3; Table S2: Ii is not clear: why stomata diameters and density were low in br, but transpiration and assimilation were increased in br? It should be clarified.
Response: Thanks for your comments. We realized our serious errors after your comments. The stomata diameters and density were measured using 3-year-old tested lines. The photosynthetic parameters were measured using 2-year-old tested lines. The stomatal characteristics could not be used to explain the transpiration and assimilation because the materials are in different years. Therefore, we have deleted the results about photosynthetic parameters.
8. P. 5, line 214: Figures 3a, b, c, d do not show WUE.
Response: Thanks for your comments. The WUE was showed in the Figure S2 in supplement previously. However, we had deleted all parts about measurement of photosynthetic parameters, including method, result and discussion. The reasons were based on the response of Point 7 and Point 9.
9. P. 5, lines 211-213; P. 15-16, lines 404-406, Figures 2c, 12a.b: Why expression of photosynthetic genes was reduced in the br, but photosynthetic assimilation of CO2 was increased in the mutant? It should be clarified.
Response: Thanks for your comments. As the transition from SAM into leaves is a complex biological process that needs internal and environmental factors involved. The expression of photosynthetic genes were obtained from transcriptome analysis of 3-year-old br apical buds and the photosynthetic assimilation of CO2 was investigated in 2-year-old br leaves. However, the expression of genes in apical buds and leaves were quite different. The expression of photosynthetic genes could not be used to explain photosynthetic assimilation of CO2 because materials are in different years and tissues. Therefore, we have deleted the results about photosynthetic parameters and DEGs related to photosynthesis.
Reviewer 2 Report
The submitted manuscript entitled: Transcriptome Analysis of a Multiple-Branches Mutant Terminal Buds in Betula platyphylla × B. Pendula presents interesting study on hybrid between two birch species linked with gene expression and leaf functional traits and photosynthetic physiology. However in current form needs substantial corrections.
The aim of study and hypothesis should be more specified.
Some formulations are unclear or incorrect, e.g.:
- agronomic traits - ???
- plant type – tree, shrub, other …?
- photosynthetic characteristics – photosynthetic rate, other?
- plant type system - ???
- biological replicates -> replicates?
- clear day - ???
- leaf color formation - ???
L. 4. And L. 83. B. Pendula – species name should be written with small letter. Moreover first time used scientific names should be given with author abbreviation.
L. 58. … can activate the activity of SAM … - …can activate SAM?
L. 60-80. This fragment is really hard to following. It must be rewritten or presented in table.
L. 110. bud morphology (stereomicroscope, Olympus) – model number should be given
L. 132. 2000(Thermo) – no space
Figures 6, 7, 8- the fonts are too small and text is blurred and unreadable.
L. 454-455. Plant hormones play a key role in plant growth and development, so we analyzed genes involved in biosynthesis, transduction and response of plant hormones. – this sentence should be replaced to Introduction.
I recommend to explain abbreviations in figure and table captions.
I’m not native speaker, but I’m think that whole manuscript must be rewritten and corrected for English before publishing in Forests. In current form the text is chaotic and not easy for following.
Author Response
Forests
April 12, 2019
Dear Reviewer，
Re: Manuscript reference No.forests-485631
Please find attached a revised version of our manuscript "Transcriptome Analysis of a Multiple-Branches Mutant Terminal Buds in Betula platyphylla × B. pendula".
Thank you very much for your comments and suggestions. We have revised the manuscript, according to the comments and suggestions of reviewers and editor, and point by point to, the comments as listed below. Revisions in the text are shown using revision mode for additions and deletions.
We hope that the revisions in the manuscript and our accompanying responses will be sufficient to make our manuscript suitable for publication in Forests.
Looking forward to hearing from you soon.
With kindest regards,
Yours Sincerely
The submitted manuscript entitled: Transcriptome Analysis of a Multiple-Branches Mutant Terminal Buds in Betula platyphylla × B. Pendula presents interesting study on hybrid between two birch species linked with gene expression and leaf functional traits and photosynthetic physiology. However in current form needs substantial corrections.
The aim of study and hypothesis should be more specified.
Response: Thanks for your comments. The research ‘highlights’ and ‘background and objectives’ has been rewritten as follows:
Research highlights: To investigate the molecular mechanism of the formation of a multiple–branches birch mutant (br), we explored genes that were genome-wide differentially expressed in main and lateral branches apical buds of br. Background and Objectives: The plant architecture not only has effects on the process of plant growth and development, but also affects agronomic characters. In woody plants, branches determine the application value of timber. Therefore, analyzing genes that were differentially expressed in br apical buds will bring new insights to understand the molecular basis of plant architecture alteration.
Some formulations are unclear or incorrect, e.g.:
- agronomic traits - ???
Response: Thanks for your comments. We have changed “agronomic traits” into “agronomic characters” in line18.
- plant type – tree, shrub, other …?
Response: Thanks for your comments. We have changed “plant type” into “plant architecture” in line16 and line20.
- photosynthetic characteristics – photosynthetic rate, other?
Response: Thanks for your comments. Based on the opinions of other reviewers, we removed photosynthesis-related results, including photosynthetic characteristics.
- plant type system - ???
Response: Thanks for your comments. We have changed “plant type” into “plant architecture” in line16 and line20.
- biological replicates -> replicates?
Response: Thanks for your comments. We have changed “biological replicates” into “independent replicates” in line 166.
- clear day - ???
Response: Thanks for your comments. Based on the opinions of other reviewers, we removed photosynthesis-related results, including “clear day”.
- leaf color formation - ???
Response: Thanks for your comments. We have deleted the formation, and the sentences had been rewritten as follows:
Mu et al. and Lin et al. performed transcriptome analysis of B. pendula ‘Dalecarlica’ and B. pendula ‘Purple Rain’ to explore the mechanism of the leaf shape and leaf color, respectively.
L. 4. And L. 83. B. Pendula – species name should be written with small letter. Moreover first time used scientific names should be given with author abbreviation.
Response: Thanks for your comments. We have changed “B. Pendula” into “B. pendula” in line4 and line96.
L. 58. … can activate the activity of SAM … - …can activate SAM?
Response: Thanks for your comments. We have changed “activate the activity of SAM” into “initiate the activity of SAM” in line 53.
L. 60-80. This fragment is really hard to following. It must be rewritten or presented in table.
Response: Thanks for your comments. We have rewritten this fragment as follows:
The formation of SAM is a crucial biological process for plant development that is determined by multiple genes, including genes involved in cell division and differentiation, hormones biosynthesis, and transduction. According to cytoplasmic density and cell division rate, SAM was divided into three zones, including peripheral zone (PZ), central zone (CZ), and rib zone (RZ) [13,14]. WUSCHEL (WUS) is critical for stem cell fate determination that specify the identity of SAM. Mutations in WUS exhibit the phenotype of misspecification of stem cells [15]. CLAVATA (CLV1, CLV2, CLV3) genes promote the progression of meristem cells toward organ initiation. There is a negative feedback regulatory loop between WUS and CLV to maintain the identity of SAM [16-19]. The class I KNOX family genes influence the differentiation of SAM via activating Isopentenyltransferase 7 (IPT7) and Arabidopsis Response Regulator 5 (ARR5) and repressing the GA 20-oxidases (GA20 ox) [20,21]. AIL genes Aintegumenta (ANT), Aintegumenta-like 6 (AIL6) and Aintegumenta-like7 (AIL7) are involved in regulating the activity of SAM. In Arabidopsis thaliana, ant, ail6 and ail7 mutations show a reduced cell division in the meristem region, resulting in plants that initiate only a few leaves prior to termination of shoot apical meristem activity [22]. In addition, microRNAs could also regulate the activity of SAM via regulating its target genes. Squamosa Promoter Binding protein-like (SPL, target gene of miR156) and APETALA2 (AP2, target gene of miR172) could activate the transition from SAM into flowers. Cup-Shaped Cotyledon (CUC, target gene of miR164) genes are required for the formation of SAM [23-26].
L. 110. bud morphology (stereomicroscope, Olympus) – model number should be given
Response: Thanks for your comments. We have added series and country of Olympus as follows: stereomicroscope SZX7 (Olympus, Tokyo, Japan) in line 126.
L. 132. 2000(Thermo) – no space
Response: Thanks for your comments. We have added space in line153.
Figures 6, 7, 8- the fonts are too small and text is blurred and unreadable.
Response: Thanks for your comments. We have changed the Figures in revised manuscript.
L. 454-455. Plant hormones play a key role in plant growth and development, so we analyzed genes involved in biosynthesis, transduction and response of plant hormones. – this sentence should be replaced to Introduction.
Response: Thanks for your comments. We have removed this sentence from discussion.
I recommend to explain abbreviations in figure and table captions.
Response: Thanks for your comments. We have added abbreviations in figure and table captions.
I’m not native speaker, but I’m think that whole manuscript must be rewritten and corrected for English before publishing in Forests. In current form the text is chaotic and not easy for following.
Response: Thanks for your comments. We have corrected some nonstandard concepts and sentences in the revised manuscript.
Reviewer 3 Report
The manuscript was describing the transcriptome analysis of main and lateral branch apical buds to find the differences of gene expression.
The article is scientifically valid; and proposed well. Even though, the paper needs to be revised linguistically as there are many typing errors and sentences to be re phrased.
Also the figure are not clear for readers, and needs to be readable.
Hereinafter some of the minor notes on the manuscript:
Line 22: "CINNAMOYL-COENZYME A REDUCTASE 1"; preferred to be in small letter as Cinnamoyl-CoA reductase 1. This is valid for many others within the manuscript such as in line (63-80)
Line 244: The Q30 percentages were all over 93.711 %. (use two digital percentage (93.71%))
Line 257: Table 3 Annotatd to be Annotated
Line 13; 225;270; 324; 347; … and in many others; the researcher have used to write (we did…). The use of passive sentences could be much better for readings
Line 294 & 318: The Figures 6 & 7 are not clear for readers.
Regards
Author Response
Forests
April 12, 2019
Dear Reviewer，
Re: Manuscript reference No.forests-485631
Please find attached a revised version of our manuscript "Transcriptome Analysis of a Multiple-Branches Mutant Terminal Buds in Betula platyphylla × B. pendula".
Thank you very much for your comments and suggestions. We have revised the manuscript, according to the comments and suggestions of reviewers and editor, and point by point to, the comments as listed below. Revisions in the text are shown using revision mode for additions and deletions.
We hope that the revisions in the manuscript and our accompanying responses will be sufficient to make our manuscript suitable for publication in Forests.
Looking forward to hearing from you soon.
With kindest regards,
Yours Sincerely
The manuscript was describing the transcriptome analysis of main and lateral branch apical buds to find the differences of gene expression.
The article is scientifically valid; and proposed well. Even though, the paper needs to be revised linguistically as there are many typing errors and sentences to be re phrased.
Response：Thanks for your comments. We have corrected the typing errors and rephrase sentences in the revised manuscript.
Also the figure are not clear for readers, and needs to be readable.
Response：Thanks for your comments. We have changed the figures to be more readable.
Line 22: "CINNAMOYL-COENZYME A REDUCTASE 1"; preferred to be in small letter as Cinnamoyl-CoA reductase 1. This is valid for many others within the manuscript such as in line (63-80)
Response: Thanks for your comments. We have changed "CINNAMOYL-COENZYME A REDUCTASE 1" into “Cinnamoyl-CoA reductase 1” in line 22. Others were also changed in line (59-72, 384-393, 459-477).
Line 244: The Q30 percentages were all over 93.711 %. (use two digital percentage (93.71%))
Response: Thanks for your comments. We have changed "93.711 %" into “93.71%” in line 2264.
Line 257: Table 3 Annotatd to be Annotated
Response: Thanks for your comments. We have changed "Annotatd" into “Annotated” in Table 3.
Line 13; 225;270; 324; 347; … and in many others; the researcher have used to write (we did…). The use of passive sentences could be much better for readings
Response: Thanks for your comments. We have changed the sentences in the revised manuscript. For example, we have changed “To know whether the phenotypes of br would recover over time, we measured the plant growth characteristics of 3-year-old WT, OE2 and br” into “To further investigate whether the phenotypes of br would recover over time, the plant growth traits of 3-year-old WT, OE2 and br were measured.” in line185.
Line 294 & 318: The Figures 6 & 7 are not clear for readers.
Response: Thanks for your comments. We have changed the Figues in revised manuscript.
Round 2
Reviewer 2 Report
The text and figure have been significantly improved. The manuscript is suitable for publication in Forests journal.
