Preliminary Classification of the ABC Transporter Family in Betula halophila and Expression Patterns in Response to Exogenous Phytohormones and Abiotic Stresses

Round 1

Reviewer 1 Report

The present work provides, for the first time, a characterization of the transcriptional response of several ABC genes to different phytohormonal signals and abiotic stressors inBetula halophila, an endangered halotolerant plant from the birch family which is endemic to China. 

 

More physiological than molecular studies have been performed in this species, and therefore the present work entail a step forward in the identification of molecular mechanisms in stress-tolerant plants. 

 

I consider, however, that the manuscript in its present form should not be accepted for publication until the following points are amended:

 

§ The English grammar must be revised throughout the manuscript.

§ This work begins with the identification of ABC transcripts from B.halophilatranscriptomic data. Such transcriptome dataset should be described (tissues, experimental conditions, microarray/RNAseq?, etc), cited, or deposited in a public database, e.g. SRA, and provide an accession number. After describing how the transcriptomic data was obtained, the authors could briefly discuss why only 15 members from one of the largest gene families in eukaryotes (with typically >100 members) were detected.

§ In the introduction, several known functions for ABC transporters are mentioned. However, some ABC-containing proteins (i.e., those form ABCF and ABCE subfamilies) seem to function in translation regulation rather than in transport. See for example a recent review (https://0-doi-org.brum.beds.ac.uk/10.3389/fpls.2018.01125) and update this aspect.

§ In relation to the previous point, 6 out of the 15 ABC proteins studied in this work showed no transmembrane domains, suggesting that these are soluble proteins more related to the functional group of the NON-transporters. This would require some discussion in this manuscript. And I suggest not to refer to these ABC proteins as “soluble transporters” (as in Figure 1 and line 33,35,128), but “soluble ABC proteins” instead.

§ In the present Table 1, the last column refers to the number of transmembrane domains (TMDs) found in the BhABC proteins. As this family of proteins is known to present a 1 or 2 TMDs with typically 6-10 alpha-helices, I wonder whether the authors indicate the number of helices rather than TMDs here. Please clarify this information.

§ In general, the first sentence of every “Results” section should motivate better the experiment that is going to be described. Please amend this aspect.

§ The resolution of Figure1 and the heatmaps in Fig3-13 must be improved to make the alphanumeric characters readable.

§ Figure 2 legend provides too little information on how the phylogeny was analyzed. The reader will thank to have at least the following under a phylogenetic tree figure: type of tree; number of homologs represented form each species (always clarify to the readers what At and Os refer to in the legend.); the clustering method used, typically neighbor-joining method; method for calculation of the evolutionary distances; what the numbers mean, bootstrap percentages?; the number of replicates.

§ Throughout the text, please refer to “expression pattern” only when comparing expression response in different tissues, and state “transcriptional response” when referring to up- or down-regulation over time.

§ Because the transcriptomic data is available to the authors, it would be nice to represent a heatmap on the expression pattern of the different genes under no treatment, by representing their relative expression in the different tissues at time 0.

§ Figure3 to 13: for clarity to the reader, I suggest framing the 4 different tissues in the heatmaps and the PCR pictures. Moreover, it would help to indicate the time of the treatment and the name of the BhABC genes also in the PCR pictures.

§  It is speculated in the discussion that genes responsive to abiotic stress might be regulated by ABA, as there are common genes responding to the hormone and the different stressors. One point that the authors could discuss here is how saline stress affect endogenous hormones in plants. See for example Šimura et al., 2018 (https://0-doi-org.brum.beds.ac.uk/10.1104/pp.18.00293), where an hormonomic approach found the levels of ABA and many other hormones affected by NaCl treatment.

 

Other minor issues:

 

§ Line 35: rephrase

§ Line 56: I suggest not to say “to understand the mechanism of BhABC regulation” but “to understand the transcriptional response of BhABC genes” instead

§ Line 67: Replace Chromium chloride with Cadmium chloride

§ Line 100: I have the feeling that some text is missing here, leaving an incomplete sentence. Anyhow, no semi-quantitative analysis based on numerical values from the band brightness is presented in this work.

§ Line 101: the last sentence fits better in 2.2.1 section.

§ To respect the order of appearance, Table 2 should be named Table 1, and vice versa.

§ Where possible, TMDs should also be depicted in Figure1

§ Line 110-113: in lack of further experimental evidence, please avoid stating that BhABCs are “localized” to certain cell structures. These are so far “predicted to be located” or "predicted to localize"

§ Figure2 and throughout the text: PDR, WBC, ATH, MDR, TAP, ATM, and MRP, as subclasses of ABC proteins should be defined in the Figure2 legend or included in the Abbreviation list, for readers not familiar with ABC proteins.

§ Heatmaps in Fig3-13: a short description of the parameters used to generate the heatmaps with HemI could be given in the methods section, or in the figure legend.

§ Line 385: the number of genes from ABCB and ABCG subfamilies responding to the treatments should be a percentage of the total. What does the interval 58-83% mean?

§ Sentence in line 392-394 is redundant with the sentence in line 397-399. Please rephrase this part of the discussion.

§  It would help the readers that the authors list the genes they refer to in lines 423 and 424, instead of saying the BhABC genes.

Author Response

Dear reviewer,
We have studied your comments carefully and made revision which marked in red in the paper. We have tried our best to revise our manuscript according to the comments. The latest revised manuscript and the pictures in the text have been uploaded in the attachment.

Response to Reviewer 1 Comments

Point 1:The English grammar must be revised throughout the manuscript.

 

Response 1: This manuscript has been revised by professional English editing.

 

Point 2: This work begins with the identification of ABC transcripts from B.halophila transcriptomic data. Such transcriptome dataset should be described (tissues, experimental conditions, microarray/RNAseq?, etc), cited, or deposited in a public database, e.g. SRA, and provide an accession number. After describing how the transcriptomic data was obtained, the authors could briefly discuss why only 15 members from one of the largest gene families in eukaryotes (with typically >100 members) were detected.

 

Response 2: We are very sorry for our negligent writing, and the description and reference were added to article 1. Introduction

 In previous study, after B. halophila had been exposed to 200 mM NaCl for 0 h or 24 h to simulate salt stress, cDNA libraries were prepared from leaves of B. halophila. Subsequently, high-throughput sequencing was conducted to identify differentially expressed genes (SRA: SRP146369). Gene expression profile analysis detected 15 differentially expressed BhABC genes in response to salt stress . Based on this result, we studied the expression profiles of these 15 genes in leaves, xylem, roots, and apical buds to better understand BhABC gene transcriptional responses to exogenous plant hormones and abiotic stressors under various conditions.

Point 3: In the introduction, several known functions for ABC transporters are mentioned. However, some ABC-containing proteins (i.e., those form ABCF and ABCE subfamilies) seem to function in translation regulation rather than in transport. See for example a recent review (https://0-doi-org.brum.beds.ac.uk/10.3389/fpls.2018.01125) and update this aspect.

 

Response 3:In the introduction, the function introduction of ABCE and ABCF family has been added.

 

Point 4: In relation to the previous point, 6 out of the 15 ABC proteins studied in this work showed no transmembrane domains, suggesting that these are soluble proteins more related to the functional group of the NON-transporters. This would require some discussion in this manuscript. And I suggest not to refer to these ABC proteins as “soluble transporters” (as in Figure 1 and line 33,35,128), but “soluble ABC proteins” instead.

 

Response 4: A description of six proteins without a transmembrane structure has been added in discussion, and “soluble transporters” have been changed to “soluble ABC proteins”according to the Reviewer’s suggestion.

 

Point 5: In the present Table 1, the last column refers to the number of transmembrane domains (TMDs) found in the BhABC proteins. As this family of proteins is known to present a 1 or 2 TMDs with typically 6-10 alpha-helices, I wonder whether the authors indicate the number of helices rather than TMDs here. Please clarify this information.

 

Response 5: We are very sorry for our negligent writing, In Table 1 (now Table 2) ，it should be the number of helices, which has been modified in the text.

 

Point 6: In general, the first sentence of every “Results” section should motivate better the experiment that is going to be described. Please amend this aspect.

 

Response 6: We have re-written the first sentence of the every Result according to the Reviewer’s suggestion. It has been added in the text.

 

Point 7: The resolution of Figure1 and the heatmaps in Fig3-13 must be improved to make the alphanumeric characters readable

 

Response 7: The resolution of Figure 1 and Fig3-13 has been improved according to the Reviewer’s suggestion.

 

Point 8: Figure 2 legend provides too little information on how the phylogeny was analyzed. The reader will thank to have at least the following under a phylogenetic tree figure: type of tree; number of homologs represented form each species (always clarify to the readers what At and Os refer to in the legend.); the clustering method used, typically neighbor-joining method; method for calculation of the evolutionary distances; what the numbers mean, bootstrap percentages?; the number of replicates.

 

Response 8:We have made correction according to the reviewer’s comments and provided the detailed description of phylogenetic tree.

The phylogenetic tree was drawn using MEGA 5.0 and was based on the following parameters: neighbor-joining tree method, complete deletion, and 500 bootstrap replicates. OsABC denotes O. sativa ABC proteins (including 24 members), AtABC denotes Arabidopsis ABC proteins (including 29 members) and BhABC denotes B. halophila ABC proteins (including 15 members). Numeric values indicate estimated evolutionary distances. This information has been added in the legend of Figure 2 and in Method 2.2.1.

 

Point 9: Throughout the text, please refer to “expression pattern” only when comparing expression response in different tissues, and state “transcriptional response” when referring to up- or down-regulation over time.

 

Response 9: We have made correction according to the reviewer’s comments. In the article, when it comes to up and down-regulation over time, the expression pattern has been changed to a transcriptional response.

 

Point 10: Because the transcriptomic data is available to the authors, it would be nice to represent a heatmap on the expression pattern of the different genes under no treatment, by representing their relative expression in the different tissues at time 0.

 

Response10: In this experiment, the expression level of 0h in different tissues is the expression level when untreated.

 

Point 11: Figure3 to 13: for clarity to the reader, I suggest framing the 4 different tissues in the heatmaps and the PCR pictures. Moreover, it would help to indicate the time of the treatment and the name of the BhABC genes also in the PCR pictures.

 

Response 11: We have made correction according to the reviewer’s comments. The treatment time has been added to the electropherogram in Figure 3 to 13, and the gene name is placed between the heat map and the electropherogram. Each tissue part has been separated by a dotted line, and the picture has been modified.

 

Point 12:  It is speculated in the discussion that genes responsive to abiotic stress might be regulated by ABA, as there are common genes responding to the hormone and the different stressors. One point that the authors could discuss here is how saline stress affect endogenous hormones in plants. See for example Šimura et al., 2018 (https://0-doi-org.brum.beds.ac.uk/10.1104/pp.18.00293), where an hormonomic approach found the levels of ABA and many other hormones affected by NaCl treatment.

 

Response 12: How does stress affect endogenous hormones have been added in the last paragraph of the discussion.---- “Under stress, the balance between various hormone levels was destroyed, which leaded to the disturbance of plant growth rhythm and the disordered of normal functional metabolism. Therefore, within a certain limit, plants could adjust their physiological functions through the change of hormone levels to adapt to the adverse external environment. ”

Other minor issues:

Point 1: Line 35: rephrase

 

Response 1: We have re-written this sentence according to the Reviewer’s suggestion, has been modified “ full transporters contain two NBDs and two TMDs, half transporters contain one NBD and one TMD, and soluble ABC proteins only contain one NBD”

 

Point 2: Line 56: I suggest not to say “to understand the mechanism of BhABC regulation” but “to understand the transcriptional response of BhABC genes” instead

 

Response 2: We have made correction according to the reviewer’s comments.

 

Point 3: Line 67: Replace Chromium chloride with Cadmium chloride

 

Response 3: We have made correction according to the reviewer’s comments.

 

Point 4: Line 100: I have the feeling that some text is missing here, leaving an incomplete sentence. Anyhow, no semi-quantitative analysis based on numerical values from the band brightness is presented in this work.

 

Response 4:We have re-written those sentences and added experimental details according to the reviewer’s comments.

The volume of each reaction product solution was adjusted to achieve a consistent concentration of internal reference gene PCR product across samples (to represent a constant number of original plant cells per final adjusted unit volume). In this way, variations in fluorescence intensity of BhABC PCR product bands, as detected during gel electrophoresis, would reflect differences in BhABC mRNA quantity in the original cells instead of differences due to variations in sample loading among gel lanes. Each standardized PCR product solution was then electrophoresed on 1% agarose gels and the intensity of each amplified fragment on the electropherogram was analyzed using Tanon Gis software to generate a number representing the relative expression level of a given gene.

 

Point 5: Line 101: the last sentence fits better in 2.2.1 section.

 

Response 5: Since this section introduces semi-quantitative data acquisition and semi-quantitative heat map production, it is not bioinformatics analysis, so it is placed in 2.2.2.

 

Point 6: To respect the order of appearance, Table 2 should be named Table 1, and vice versa.

 

Response 6: We are very sorry for our negligent writing,The naming of Table 1 and Table 2 has been modified.

 

Point 7: Where possible, TMDs should also be depicted in Figure1

 

Response 7: Because TMD is very conservative, it is impossible to accurately predict TMDs.

 

Point 8: Line 110-113: in lack of further experimental evidence, please avoid stating that BhABCs are “localized” to certain cell structures. These are so far “predicted to be located” or "predicted to localize"

 

Response 8: We have made correction according to the reviewer’s comments.It has been corrected as “predicted to be located”.

 

Point 9: Figure2 and throughout the text: PDR, WBC, ATH, MDR, TAP, ATM, and MRP, as subclasses of ABC proteins should be defined in the Figure2 legend or included in the Abbreviation list, for readers not familiar with ABC proteins.

 

Response 9: The full name of PDR, WBC, ATH, MDR, TAP, ATM, MRP has been added to the abbreviated list.

 

Point 10: Heatmaps in Fig3-13: a short description of the parameters used to generate the heatmaps with HemI could be given in the methods section, or in the figure legend.

 

Response 10: The description of the generated heat map has been added to the last sentence of 2.2.2.----heat maps were generated using HemI v1.0 software based on the following parameter selections: linear normalized intensity data, average linkage clustering method, and similarity metric of Euclidean distance.

 

Point 11: Line 385: the number of genes from ABCB and ABCG subfamilies responding to the treatments should be a percentage of the total. What does the interval 58-83% mean?

 

Response 11:We are very sorry for our negligent writing. This ratio is not a good illustration of the problem, this sentence has been changed to“Of the genes responding to these stimuli, a high proportion belonged to ABCB and ABCG subfamilies, which happen to be the same plant subfamilies currently under study for their suspected roles in exogenous hormone and abiotic stressor responses ”

 

Point 12: Sentence in line 392-394 is redundant with the sentence in line 397-399. Please rephrase this part of the discussion.

 

Response 12: We have made correction according to the reviewer’s comments.The extra sentences have been deleted.

 

Point 13:  It would help the readers that the authors list the genes they refer to in lines 423 and 424, instead of saying the BhABC genes.

 

Response 13:Sorry, we are unable to find the issue from lines 423-424.

 

Author Response File: Author Response.docx

Reviewer 2 Report

I would like to know the transcriptome method. RNA-seq, microarray or others? 

The characters in figures except for Fig.2 are too small. I could not judge the validity.

Promoter sequence analysis should be performed.

Please confirm the concentration of the hormone treatments in the method section. For example, the concentration of ABA is 100 mol / L. 

Author Response

Dear reviewer,
We have studied your comments carefully and made revision which marked in red in the paper. We have tried our best to revise our manuscript according to the comments. The latest revised manuscript have been uploaded in the attachment.

 

Response to Reviewer 2 Comments

Point 1:I would like to know the transcriptome method. RNA-seq, microarray or others? 

 

Response 1:the transcriptome method is RNA-seq,This part of content has been add in 1. Introduction.

 

Point 2:The characters in figures except for Fig.2 are too small. I could not judge the validity.

 

Response 2:The sharpness of the pictures in this article has been modified according to the reviewer’s comments.

 

Point 3:Promoter sequence analysis should be performed.

 

Response 3: Thank the reviewers for their suggestions, because the Betula halophila genome has not yet been sequenced, the promoter sequences of ABC protein genes are not available yet, but we'll do it later.

 

Point 4:Please confirm the concentration of the hormone treatments in the method section. For example, the concentration of ABA is 100 mol / L.  

 

Response 4: We are very sorry for our negligent writing. Hormone concentrations have been confirmed again, 25 mg/L gibberellin (GA3), 1 μmol/L methyl jasmonate (MeJA), 350 μmol/L salicylic acid (SA), 0.2 mg/L brassinolide (BR), 50 mg/L cytokinin (6-BA), 100μmol/L abscisic acid (ABA), or 50mg/L auxin (IAA).

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Just a final minor issue

Figures 3-13: the indication of “left” and “right” in the legend should be changed according to the new position of the gel pic and the heatmap.

Author Response

Response to Reviewer 1 Comments

Point 1:Figures 3-13: the indication of “left” and “right” in the legend should be changed according to the new position of the gel pic and the heatmap.

Response 1:We are very sorry for our negligent writing, in figures 3-13， the indication of “left” and “right” in the legend have changed.

The latest version of the manuscript has been uploaded to the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript has been significantly improved.

Author Response

Dear reviewer,

The latest version of the manuscript has been uploaded to the attachment.

Author Response File: Author Response.docx