Genome-Wide Identification and Transcriptional Expression Profiles of the F-box Gene Family in Common Walnut (Juglans regia L.)

Round 1

Reviewer 1 Report

The main purpose of this manuscript was to identify and characterize the F-box family of genes from common walnut, a gene family with roles in a wide variety of plant processes.  They used F-box sequences from Arabidopsis to search the common walnut genome for F-box-like genes and identified 74 candidate genes.  They characterized these genes into three groups and eight subfamilies and mapped them onto the putative chromosomes.  They also examined the gene structure and protein domains present in each F-box candidate.  In order to gain more information into the potential roles of these genes, the authors compared their expression in publicly available gene sets of vegetative tissues and generated their own transcriptomes from male and female flowers collected in April and May.  They found that the F-box genes had three general patterns of expression, with one group showing enrichment in floral samples as compared to vegetative samples.  Comparing expression profiles of F-box genes between tissues showed that male and female flowers had more similar expression of F-box genes than any other paired sample set shown.  The authors concluded that members of the F-box family in walnut may play an important role in flowering in common walnut.  This paper illustrates how a gene family can be analyzed in detail from a species of interest.  However, it has concerns that need addressing. 

 

Main concerns

 

1.       A main concern for this paper is the scientific approach.  Some of the methods and analysis are lacking important details required to assess the validity and rigor of the approach.  In particular, the authors need to detail exactly how samples were collected (which trees, how many flowers, how/if samples were pooled), and what constituted their biological and technical replicates.  Also, why were there differing numbers of male and female samples?  It is unclear how the predicted F-box protein sequences of walnut were identified.  Were they from an existing dataset or predicted as a part of this manuscript?  How were the expression data in Figure 8 obtained?  QPCR?  If so, please add this information to the methods section along with a supplemental file of the primer sequences. 

 

2.       The manuscript would be improved by a stronger tie to tree data.  The introduction provided detailed information on F-box families in model and crop species.  While there is a wealth of information on these species, they are not closely related to trees.  It would be more informative to focus on comparisons with other trees species.  The authors did include a brief mention of apple and pear and ought to expand on this theme and add other species.  Populus has been characterized in this regard (please refer to the manuscript titled The F-Box Gene Family Is Expanded in Herbaceous Annual Plants Relative to Woody Perennial Plants) and has fewer F-box genes that herbaceous annuals and is a better comparator than Arabidopsis. 

 

3.       The paper needs to be strengthened by making connections between the gene expression data and the biology of walnut.  In particular, the developmental stages of the floral samples needs to be described (and ideally photographed and included as a figure) and tied back to the F-box data.  F-box proteins are important for floral processes, such as organogenesis and pollen tube regulation.  By determining which F-box genes are upregulated at different floral stages the authors could better predict the roles of said genes. 

 

4.       Some of the analysis is rather speculative, in particular, the GO annotation analysis and the network of miRNAs and putative F-box targets.  The former is unnecessary as the F-box itself is a defining feature and gives a predicted function.  To say that 41 of the 74 predicted F-box proteins have “molecular function” is not descriptive.  Either more extensive analysis is needed or this section can be reduced.  The predicted network presented in figure 4 is speculative as there is no data presented for the expression of the miRNAs.  Perhaps this figure is best omitted. 

 

Minor concerns

 

5.       There was a huge variance in the coding region size and structure of the F-box gene family.  How does this variability compare to other F-box families, especially of trees?

 

6.       Like many trees, walnuts are highly heterogeneous.  Would these inter and intra genome diversity be a confounding factor when using different trees for expression data?  Could the heterozygocity also be a complication in distinguishing duplicated genes versus alleles? 

 

7.       The link for the reference to the walnut genome (line 269) is actually for the pear genome.  Please correct this mistake. 

 

8.       The manuscript needs extensive language edits, including fixing distracting spelling errors, such as feamale instead of female in Figure 6 and elsewhere

 

9.       The text in many of the figures (such as 1, 2, 3) is so small as to be nearly unreadable, please increase the size

 

10.   Table 1 seems better suited as a supplemental file, please move it to this location

 

11.   The journal names need to be checked for formatting consistency

 

12.   It was very good to see a heatmap that was not red/green as such a color combination is not distinguishable for colorblind readers.  Please consider not using red/green as indicator colors in other figures. 

Author Response

Response to Reviewer 1 Comments

 

Thank you for your valuable comments. We have studied the valuable comments from you carefully, made a significant effort to make the work clearer, and tried our best to revise the manuscript. The point to point responds to the reviewer’s comments as following:

 

The main purpose of this manuscript was to identify and characterize the F-box family of genes from common walnut, a gene family with roles in a wide variety of plant processes.  They used F-box sequences from Arabidopsis to search the common walnut genome for F-box-like genes and identified 74 candidate genes.  They characterized these genes into three groups and eight subfamilies and mapped them onto the putative chromosomes.  They also examined the gene structure and protein domains present in each F-box candidate.  In order to gain more information into the potential roles of these genes, the authors compared their expression in publicly available gene sets of vegetative tissues and generated their own transcriptomes from male and female flowers collected in April and May.  They found that the F-box genes had three general patterns of expression, with one group showing enrichment in floral samples as compared to vegetative samples.  Comparing expression profiles of F-box genes between tissues showed that male and female flowers had more similar expression of F-box genes than any other paired sample set shown.  The authors concluded that members of the F-box family in walnut may play an important role in flowering in common walnut.  This paper illustrates how a gene family can be analyzed in detail from a species of interest.  However, it has concerns that need addressing. 

Main concerns

Point 1:  A main concern for this paper is the scientific approach.  Some of the methods and analysis are lacking important details required to assess the validity and rigor of the approach.  In particular, the authors need to detail exactly how samples were collected (which trees, how many flowers, how/if samples were pooled), and what constituted their biological and technical replicates.  Also, why were there differing numbers of male and female samples?  It is unclear how the predicted F-box protein sequences of walnut were identified.  Were they from an existing dataset or predicted as a part of this manuscript?  How were the expression data in Figure 8 obtained?  QPCR?  If so, please add this information to the methods section along with a supplemental file of the primer sequences.  

Response: Thank you for your comments. We revised the details of sample collection in “Material and Methods” carefully following our experiments and field works from Line 227 to 240 as following:

To assess the expression of common walnut F-box genes, we sampled a total of 12 fresh flowers, specifically 9 fresh female flowers and 3 male flowers from common walnut individual trees which were grown on the Qinling Mountains, collected at different development times (Table S1). For each female flower development time, we collected female inflorescence as 3, 3, 2, and 1 biological replicate from an adult tree growing on the Qinling Mountains on April 10, April 15, April 22, and May 1, respectively (Table S1). For each male flower development time, we collected one male inflorescence from an adult tree growing on the Qinling Mountains on April 10, April 11, and May 2, respectively (for details on flower tissues collection, see Table S1). We put the fresh female flowers and male flowers in liquid nitrogen prior to storage at –80℃ until use [38] (Table S1). We also added the collection date for female and male flowers collections.  The female inflorescence has one to three flowers, while male inflorescence had many small flowers. So, we collected different number samples for female and male flowers. We pooled small male flowers in this study. We also added the picture for both female and male flowers in Figure 5 in the revision.

 

We have revised the Materials and Methods section as following from Line 97-98:

The whole protein sequences of common walnut were downloaded from the NCBI database (https://treegenesdb.org/FTP/Genomes/Jure/v1.4/annotation/). After obtaining the whole common walnut protein data, we used blastp program with the reported F-box protein sequence of Arabidopsis as the queries to identify F-box gene family of common walnut, so we wrote in the results section that we identified the protein sequence of common walnut F-box gene family with 74 members.

“The genome sequences of J. regia were downloaded from the common walnut gene database (https://www.ncbi.nlm.nih.gov/genome/17683)” to “the whole protein sequences of common walnut were downloaded from NCBI database(https://www.ncbi.nlm.nih.gov/genome/17683)”.

 

In the Materials and Methods section,

We quantified these gene expression levels based on their fragments per kilobase of exon per million reads mapped (FPKM) values using Cufflinks with default parameters [52]. The expression data in Figure7 were obtained from transcriptome expression profiles, which based on FPKM values using by error bar (Table S5, Figure 7) in the revision. We also added the sentences from Line 253 to 255 as following: “For the expression level of several F-box genes with error bar, the ggplot2 R package was used [55]. ”

 

We added the legend for Figure7 from line 467 to 468 as following: “The expression level data are as shown in Table S5. We used 3 or 2 biological replicates from the same organization in the same period in Table S5.”

 

Point 2: The manuscript would be improved by a stronger tie to tree data.  The introduction provided detailed information on F-box families in model and crop species.  While there is a wealth of information on these species, they are not closely related to trees.  It would be more informative to focus on comparisons with other trees species.  The authors did include a brief mention of apple and pear and ought to expand on this theme and add other species.  Populus has been characterized in this regard (please refer to the manuscript titled The F-Box Gene Family Is Expanded in Herbaceous Annual Plants Relative to Woody Perennial Plants) and has fewer F-box genes that herbaceous annuals and is a better comparator than Arabidopsis. 

Response: Thank you for your valuable comments. We found a total of six articles focused on the wood plants F-box genes in previous study. So, we revised the DISSCUSSION section and INTRODUCTION section carefully in the revision.

 

We added some sentences in the DISSCUSSION section as followings from line 653 to 667 as followings: “Some previous studies have shown that F-box genes and mutants play an important role in the development of pollen tubes in woody plants. In apples, the F-box gene was named SFBB, which is specifically expressed in pollen tubes. In Rosaceae plants, such as sweet cherry and Japanese, the F-box gene was named SFB and plays an important role in the process leading to self-incompatibility. SLF in Prunus mume was also confirmed to be specifically expressed in pollen tubes by cloning the F-box gene. Alleles SFB1, SFB2, SFB4, and SFB5 in sweet cherry proved to play a self-incompatible effect by affecting the development of pollen tubes. Allele S4sm, lacking the SFBB1-S4 protein, caused no significant effect on pollen tube, while the S5 haplotype encoded SFBB1 protein can cause pistils to accept other genes [29,35-40], so there is strong evidence that the F-box gene is relevant in pollen development. In this study, the results showed that some genes (Jure 07950.t1, Jure 21178.t1, Jure 11456.t1, Jure 07156.t1, Jure 07788.t1, Jure 21675.t1, Jure 19792.t1, Jure 05249.t1, Jure 26563.t1, Jure 20019.t1, Jure 14341.t1, and Jure 26807.t1) were expressed highly in female flowers, suggesting that the F-box gene family of common walnut was the vital genes regulating the flowering (Figures 4–6).”

 

We added some sentences in the INTRODUCTION section from Line 64-66 as followings: “Important crops contain F-box genes, too—for example, rice, maize, soybean, and chickpea contain 678, 509, 359, and 285 F-box genes, respectively [25-28].”   We also added some sentences in the INTRODUCTION section as followings from Line 80 to 96. “In apple (Malus domestica) and Japanese pear (Pyrus pyrifolia), since each S haplotype contains two or three related genes, the genes were named SFBB for S locus F-box brothers. The SFBB genes were specifically expressed in pollen, and variable regions of the SFBB genes were under positive selection [32]. Many Prunus species, including sweet cherry and Japanese apricot, of the Rosaceae, display an S-RNase-based gametophytic self-incompatibility (GSI). An F-box protein is encoded by a gene located in the S locus region, named SFB, and it was found that there were two self-compatible (SC) haplotypes, S4¢ and Sf of Prunus, which caused the pollen-part mutant (PPM). The Sf of Japanese apricot was also considered to be a PPM. Both of them were examined and it was found that SFB was the pollen S gene in GSI in Prunus [33]; an F-box gene named SLF (S-locus F-box) was specifically expressed in pollen, but not in the styles or leaves [34]. Four additional alleles (SFB1, SFB2, SFB4, and SFB5) were cloned from sweet cherry (P. avium) and were examined, being found to have the function of allele specificity of the GSI reaction [35]. In Japanese pear (Pyrus pyrifolia), the mutant haplotype S^4sm lacked SFBB1^-S4, which caused the pollen to be rejected by pistils with an otherwise compatible S1, while it was accepted by other non-self pistils, and he S5 haplotype encoded a truncated SFBB1 protein, even though S5 pollen was accepted normally by pistils with S1 and other non-self haplotypes [36]. "

 

 

Point 3: The paper needs to be strengthened by making connections between the gene expression data and the biology of walnut.  In particular, the developmental stages of the floral samples needs to be described (and ideally photographed and included as a figure) and tied back to the F-box data.  F-box proteins are important for floral processes, such as organogenesis and pollen tube regulation.  By determining which F-box genes are upregulated at different floral stages the authors could better predict the roles of said genes.

Response: Thank you for your comments. We added some sentences in the RESULTS section from Line 372 to 378 as following:

“For female and male flowers, we collected 9 periods of female flowers and 3 periods of male flowers; the period from April to May was a key period of flower development for the flowering of common walnut, so we collected these tissues. Of course, we could notice that the members of the walnut F-box gene family were not much different within the period. The difference was that there was no obvious increase in the expression level with the developmental period.”    We also added some sentences in the RESULTS section from Line 394 to 407 as following: During the female flower ripening, it was shown that some genes had an increased expression level with female growing, for example, Jure_21178.t1, Jure_11456.t1, Jure_07156.t1, Jure_07788.t1, Jure_14341.t1, Jure_21675.t1, Jure_06725.t1, Jure_19792.t1, Jure_05249.t1, Jure_26563.t1, Jure_13358.t1, and Jure_16679.t1. In male flowers, Jure_10126.t1, Jure_30338.t1, Jure_26563.t1, and Jure_07156.t1 had a higher expression level on April 10 than in the other two periods. Overall, in the reproductive tissues (female and female flowers) and vegetative tissues (vegetative-bud, embryo, somatic embryo, leaves, young leaf, immature hull, hull cortex, hull peel, hull dehiscing, and root), a total of 13 members (Jure_07950.t1, Jure_21178.t1, ,Jure_07156.t1, Jure_07788.t1, Jure_14341.t1, Jure_21675.t1, Jure_06725.t1, Jure_19792.t1, Jure_06252.t1, Jure_02219.t1,Jure_05249.t1, Jure_26563.t1) of the F-box gene family were highly expressed in reproductive tissues, while those in other vegetative tissues were low in expression, and 3 F-box genes (Jure_29522.t1,Jure_10126.t1,Jure_30338.t1) were higher in reproductive tissues than in nutritive tissues (Figure 4).   We added some sentences in the RESULTS section from line 434 to 437 as following: “In order to compare the differences between the F-box gene expression level in reproductive tissues and vegetative tissues, we selected male and female flowers, young leaves, and hull with biological replicates to create a clear expression pattern as shown in Figure 5A.”  We also added some sentences in the RESULTS section as followings from Line 446 to 451 as following:“ There was no significant positive or negative correlation between the expression level of the F-box gene in leaves and the expression level in female flowers; this was also the case in hull and female flowers, leaves and male flowers, and hull and male flowers. We averaged the expression values of vegetative and reproductive tissues at various stages and performed regression analysis, which did not show an obvious positive or negative correlation.”   

 

We also added some sentences in the DISSCUSSION section as followings from line 623 to 630 as following: During the critical development period of walnuts, from April to May, there was no increase in the expression of the F-box gene family between walnuts, regardless of the period of the female flower, or the male flower. In the three periods, only different genes have different expression levels, but they are not different at different times. Therefore, we speculate that this is because the walnut F-box gene family played an important role in the flowering and development process, so the key genes had been expressed throughout the flowering period, so there was no difference in the developmental stage, making previously reported research results are consistent (Figure 4).”

The conclusion as “During female flower ripening, it was shown that some genes exhibited an increased expression level with the female growing. Overall, in the reproductive tissues and vegetative tissues, a total of 13 members(Jure_07950.t1, Jure_21178.t1, ,Jure_07156.t1, Jure_07788.t1, Jure_14341.t1, Jure_21675.t1, Jure_06725.t1, Jure_19792.t1, Jure_06252.t1, Jure_02219.t1,Jure_05249.t1, Jure_26563.t1) of the F-box gene family were highly expressed in reproductive tissues, while those in other vegetative tissues were low in expression, and 3 F-box genes(Jure_29522.t1,Jure_10126.t1,Jure_30338.t1) were higher in reproductive tissues than in nutritive tissues.” were also added into the CLOUSION section from Line 678 to 683 in the revision.

 

Point 4: Some of the analysis is rather speculative, in particular, the GO annotation analysis and the network of miRNAs and putative F-box targets.  The former is unnecessary as the F-box itself is a defining feature and gives a predicted function.  To say that 41 of the 74 predicted F-box proteins have “molecular function” is not descriptive.  Either more extensive analysis is needed or this section can be reduced.  The predicted network presented in figure 4 is speculative as there is no data presented for the expression of the miRNAs.  Perhaps this figure is best omitted. 

 

Response: Thank you for your comments.

We omitted the GO annotations analysis and Figure4 in the Materials and Methods section.

We deleted the words “GO term” from Line 172 in the Materials and Methods section.

We deleted the sentence from Line 174 to 175 in the Materials and Methods section as followings:

“GO annotations for F-box gene family were retrieved from the common walnut genome project (http://peargenome.njau.edu.cn/)”

We deleted the sentence from Line 188 to 197 in the Materials and Methods section. We deleted the phrase “GO and” from line 335 in the RESULTS section.

We deleted the sentence from Line 337 to 344 in the RESULTS section

We revised the sentence from Line 356 in the RESULTS section as floowings:

“3.4. Expression profile analysis of walnut F-box genes”

We deleted the sentence from Line 357 to 367 in the RESULTS section.

We deleted the sentence from Line 594 to 602 in the DISCUSSION section.

We deleted the Figure 4 and Supplementary Table S4.

Minor concerns

 

Point 5: There was a huge variance in the coding region size and structure of the F-box gene family.  How does this variability compare to other F-box families, especially of trees?

Response: Thank you for your comments. The present study identified 74 F-box genes from common walnut (Figure 1 Table S2), compared to the number of the higher plants such as Arabidopsis, M. truncatula (972), maize (359) and rice (687) had less F-box genes in their genomes [23,25,26,31]. In perennial woody plants such as populus trichocarpa, apple, and pear contained 320, 517, and 226 F-box genes, respectively [24,29,30]. The number of F-box gens of woody plants and herbaceous annual plants (Arabidopsis thaliana, rice, maize, and soybean) had of this gene family members [24-27,29,30]. The highly variations of F-box genes in woody plant species let we cannot compare the gene structure relative to walnut and other trees. However, in the previous study, the authors did not show gene structure of populus trichocarpa, apple, and pear [24,29,30]. We revised the sentences in the INTRODUCTION section from Line 67 to 72 and section in the revision as following: “as well as perennial woody plants, such as populus trichocarpa, apple, and pear which contain 320, 517, and 226 F-box genes, respectively [24,29,30]. The number of F-box genes of woody plants and herbaceous annual plants (Arabidopsis thaliana, rice, maize, and soybean) has high variations [24-27,29,30].”

Point 6:  Like many trees, walnuts are highly heterogeneous. Would these inter and intra genome diversity be a confounding factor when using different trees for expression data?  Could the heterozygocity also be a complication in distinguishing duplicated genes versus alleles?

Response: Thank you for your comments. The heterozygocity of common walnut is very high cause by the wind dispersal pollination. It should be effect on the alleles when we do microsatellite PCR experiments or other nuclear sequencing data. In this study, we download the common walnut reference genome from NCBI database (https://www.ncbi.nlm.nih.gov/genome/17683). So, when we did the RNA expressions, the reference genome sequence data will be propelling principle for the evaluated the level of different tissue expression. The F-box genes were also identified by BLASTP based on the whole reference genome sequence. So, we speculate our expression data would not affect by the heterozygocity. We also did the duplicated gene analysis based on the annotated reference walnut genome sequence.\

Point 7: The link for the reference to the walnut genome (line 269) is actually for the pear genome.  Please correct this mistake. 

 Response: Thank you for your comments. We deleted the link from Line 159 to 160.

 

Point 8:   The manuscript needs extensive language edits, including fixing distracting spelling errors, such as feamale instead of female in Figure 6 and elsewhere

 Response: Thank you for your comments. We revised the words “feamale” to “female” in Figure5 in the revision.

 

Point 9:  The text in many of the figures (such as 1, 2, 3) is so small as to be nearly unreadable, please increase the size

Response: Thank you for your comments. We redo and increase the pixel as 450 dpi for figures (such as 1, 2, 3) in the revision.

 

Point 10: Table 1 seems better suited as a supplemental file, please move it to this location

Response: Thank you for your comments. We added the Table 1 as a supplemental file2.

Point 11:  The journal names need to be checked for formatting consistency

Response: Thank you for your comments. We revised the reference journal name, format, and other reference carefully in the revision in Line709, 718, 723, 732, 744, 748, 750, 766, 785, 844, and 871, respectively. We also checked our cited in the main text, too.

 

Point 12:  It was very good to see a heatmap that was not red/green as such a color combination is not distinguishable for colorblind readers.  Please consider not using red/green as indicator colors in other figures

Response: Thank you for your comments. We revised the Figures which not using red/green as indicator colors in other figures (Figure 1, Figure 2, and Figure 3).

 

We revised and edited our whole manuscript improved grammatical errors and wrongly spelled words by a professional and native English language editing company MDPI.

 

Thank you for your consideration our manuscript of “Genome-wide identification and transcriptional expression profiles of the F-box gene family in common walnut (Juglans regia L.)" publish on Journal of Forests.

 

Sincerely,

 

Zhao Peng, Ph.D

 

Author Response File: Author Response.doc

Reviewer 2 Report

The authors did an overall interesting approach and a lot of analyses. With common walnut, they used an economically important species.

Nevertheless, I like to give only a short review, because the manuscript is really hard to read because of the language. I sometimes not even understand what the authors want to say.

 

Some major concerns are:

The results section is just confusing and need a clearer red line.

The discussion mainly based on the comparison of the findings of the authors with herbaceous plants. That there are a lot of differences is obvious. A comparison with other woody plants would make much more sense.

The authors conclude only that there is a positive correlation between female and male flowers. They did such a big amount of analyses and reduce them to one statement.

Thus, my impression is that the authors did a lot of analyses but missed to combine them in a proper way. And thereby lost some of their – interesting – results.

 

I suggest Table 1 as supplementary material

Author Response

Response to Reviewer 2 Comments

 

Thank you for your valuable comments. We have studied the valuable comments from you carefully, made a significant effort to make the work clearer, and tried our best to revise the manuscript. The point to point responds to the reviewer’s comments as following:

 

The authors did an overall interesting approach and a lot of analyses. With common walnut, they used an economically important species. Nevertheless, I like to give only a short review, because the manuscript is hard to read because of the language. I sometimes not even understand what the authors want to say.

Some major concerns are:

Thank you for your valuable comments. We revised and edited the whole manuscript improved grammatical errors and wrongly spelled words by a professional and native English language editing company MDPI.

 

Point 1:  The results section is just confusing and need a clearer red line.

Response: Thank you for your comments. We added some sentences in the RESULTS section from Line 372 to 378 as following:

“For female and male flowers, we collected 9 periods of female flowers and 3 periods of male flowers; the period from April to May was a key period of flower development for the flowering of common walnut, so we collected these tissues. Of course, we could notice that the members of the walnut F-box gene family were not much different within the period. The difference was that there was no obvious increase in the expression level with the developmental period.”    We also added some sentences in the RESULTS section from Line 394 to 407 as following: During the female flower ripening, it was shown that some genes had an increased expression level with female growing, for example, Jure_21178.t1, Jure_11456.t1, Jure_07156.t1, Jure_07788.t1, Jure_14341.t1, Jure_21675.t1, Jure_06725.t1, Jure_19792.t1, Jure_05249.t1, Jure_26563.t1, Jure_13358.t1, and Jure_16679.t1. In male flowers, Jure_10126.t1, Jure_30338.t1, Jure_26563.t1, and Jure_07156.t1 had a higher expression level on April 10 than in the other two periods. Overall, in the reproductive tissues (female and female flowers) and vegetative tissues (vegetative-bud, embryo, somatic embryo, leaves, young leaf, immature hull, hull cortex, hull peel, hull dehiscing, and root), a total of 13 members (Jure_07950.t1, Jure_21178.t1, ,Jure_07156.t1, Jure_07788.t1, Jure_14341.t1, Jure_21675.t1, Jure_06725.t1, Jure_19792.t1, Jure_06252.t1, Jure_02219.t1,Jure_05249.t1, Jure_26563.t1) of the F-box gene family were highly expressed in reproductive tissues, while those in other vegetative tissues were low in expression, and 3 F-box genes (Jure_29522.t1,Jure_10126.t1,Jure_30338.t1) were higher in reproductive tissues than in nutritive tissues (Figure 4).   We added some sentences in the RESULTS section from line 434 to 437 as following: “In order to compare the differences between the F-box gene expression level in reproductive tissues and vegetative tissues, we selected male and female flowers, young leaves, and hull with biological replicates to create a clear expression pattern as shown in Figure 5A.”  We also added some sentences in the RESULTS section as followings from line 446 to 451 as following:“ There was no significant positive or negative correlation between the expression level of the F-box gene in leaves and the expression level in female flowers; this was also the case in hull and female flowers, leaves and male flowers, and hull and male flowers. We averaged the expression values of vegetative and reproductive tissues at various stages and performed regression analysis, which did not show an obvious positive or negative correlation.”   

We deleted the phrase “GO and” from line 335 in the RESULTS section.

We deleted the sentence from Line 337 to 344 in the RESULTS section

We revised the sentence from Line 356 in the RESULTS section as floowings:

“3.4. Expression profile analysis of walnut F-box genes”

We deleted the sentence from Line 357 to 367 in the RESULTS section.

 

Point 2: The discussion mainly based on the comparison of the findings of the authors with herbaceous plants. That there are a lot of differences is obvious. A comparison with other woody plants would make much more sense.

Response: Thank you for your valuable comments. We found a total of six articles focused on the wood plants F-box genes in previous study. So, we revised the DISSCUSSION section and INTRODUCTION section carefully in the revision.

 

We added some sentences in the DISSCUSSION section as followings from line 653 to 667 as followings: “Some previous studies have shown that F-box genes and mutants play an important role in the development of pollen tubes in woody plants. In apples, the F-box gene was named SFBB, which is specifically expressed in pollen tubes. In Rosaceae plants, such as sweet cherry and Japanese, the F-box gene was named SFB and plays an important role in the process leading to self-incompatibility. SLF in Prunus mume was also confirmed to be specifically expressed in pollen tubes by cloning the F-box gene. Alleles SFB1, SFB2, SFB4, and SFB5 in sweet cherry proved to play a self-incompatible effect by affecting the development of pollen tubes. Allele S4sm, lacking the SFBB1-S4 protein, caused no significant effect on pollen tube, while the S5 haplotype encoded SFBB1 protein can cause pistils to accept other genes [29,35-40], so there is strong evidence that the F-box gene is relevant in pollen development. In this study, the results showed that some genes (Jure 07950.t1, Jure 21178.t1, Jure 11456.t1, Jure 07156.t1, Jure 07788.t1, Jure 21675.t1, Jure 19792.t1, Jure 05249.t1, Jure 26563.t1, Jure 20019.t1, Jure 14341.t1, and Jure 26807.t1) were expressed highly in female flowers, suggesting that the F-box gene family of common walnut was the vital genes regulating the flowering (Figures 4–6).”

 

We added some sentences in the INTRODUCTION section from Line 64-66 as followings: “Important crops contain F-box genes, too—for example, rice, maize, soybean, and chickpea contain 678, 509, 359, and 285 F-box genes, respectively [25-28].”   We also added some sentences in the INTRODUCTION section as followings from Line 80 to 96. “In apple (Malus domestica) and Japanese pear (Pyrus pyrifolia), since each S haplotype contains two or three related genes, the genes were named SFBB for S locus F-box brothers. The SFBB genes were specifically expressed in pollen, and variable regions of the SFBB genes were under positive selection [32]. Many Prunus species, including sweet cherry and Japanese apricot, of the Rosaceae, display an S-RNase-based gametophytic self-incompatibility (GSI). An F-box protein is encoded by a gene located in the S locus region, named SFB, and it was found that there were two self-compatible (SC) haplotypes, S4¢ and Sf of Prunus, which caused the pollen-part mutant (PPM). The Sf of Japanese apricot was also considered to be a PPM. Both of them were examined and it was found that SFB was the pollen S gene in GSI in Prunus [33]; an F-box gene named SLF (S-locus F-box) was specifically expressed in pollen, but not in the styles or leaves [34]. Four additional alleles (SFB1, SFB2, SFB4, and SFB5) were cloned from sweet cherry (P. avium) and were examined, being found to have the function of allele specificity of the GSI reaction [35]. In Japanese pear (Pyrus pyrifolia), the mutant haplotype S^4sm lacked SFBB1^-S4, which caused the pollen to be rejected by pistils with an otherwise compatible S1, while it was accepted by other non-self pistils, and he S5 haplotype encoded a truncated SFBB1 protein, even though S5 pollen was accepted normally by pistils with S1 and other non-self haplotypes [36]. "

 

Point 3: The authors conclude only that there is a positive correlation between female and male flowers. They did such a big amount of analyses and reduce them to one statement. Thus, my impression is that the authors did a lot of analyses but missed to combine them in a proper way. And thereby lost some of their – interesting – results.

Response: Thank you for your comments. We also added some sentences in the RESULTS section as followings from Line 446 to 451 as following:“ There was no significant positive or negative correlation between the expression level of the F-box gene in leaves and the expression level in female flowers; this was also the case in hull and female flowers, leaves and male flowers, and hull and male flowers. We averaged the expression values of vegetative and reproductive tissues at various stages and performed regression analysis, which did not show an obvious positive or negative correlation.”   

 

We also added some sentences in the DISSCUSSION section as followings from line 623 to 630 as following: During the critical development period of walnuts, from April to May, there was no increase in the expression of the F-box gene family between walnuts, regardless of the period of the female flower, or the male flower. In the three periods, only different genes have different expression levels, but they are not different at different times. Therefore, we speculate that this is because the walnut F-box gene family played an important role in the flowering and development process, so the key genes had been expressed throughout the flowering period, so there was no difference in the developmental stage, making previously reported research results are consistent (Figure 4).”

The conclusion as “During female flower ripening, it was shown that some genes exhibited an increased expression level with the female growing. Overall, in the reproductive tissues and vegetative tissues, a total of 13 members(Jure_07950.t1, Jure_21178.t1, ,Jure_07156.t1, Jure_07788.t1, Jure_14341.t1, Jure_21675.t1, Jure_06725.t1, Jure_19792.t1, Jure_06252.t1, Jure_02219.t1,Jure_05249.t1, Jure_26563.t1) of the F-box gene family were highly expressed in reproductive tissues, while those in other vegetative tissues were low in expression, and 3 F-box genes(Jure_29522.t1,Jure_10126.t1,Jure_30338.t1) were higher in reproductive tissues than in nutritive tissues.” were also added into the CLOUSION section from Line 678 to 683 in the revision.

 

Point 4: I suggest Table 1 as supplementary material

Response: Thank you for your comments. We added the Table 1 as a supplemental file2.

 

We submitted the manuscript to MDPI for editing of English language and style.

 

Thank you for your consideration our manuscript of “Genome-wide identification and transcriptional expression profiles of the F-box gene family in common walnut (Juglans regia L.)" publish on Journal of Forests.

 

Sincerely,

 

Zhao Peng, Ph.D

 

Author Response File: Author Response.doc

Round 2

Reviewer 1 Report

Thank you for the numerous revisions to your manuscript.  I have a few suggestions to streamline the paper and highlight the link between the bioinformatics and the biology. 

 

The introduction cites a very large number of works regarding F-box genes in plants.  This section could be trimmed to better focus on the research most related to this manuscript. 

 

The sample collection was better described but could use additional clarification.  Do walnut trees show synchronized floral opening such that flowers collected on very close days represent different floral ages?  Were flowers collected from equivalent positions on each tree?  Were the trees of similar ages?  Do the large clusters of flowers (as seen in the male) have synchronized development in each cluster? 

 

One area that must be improved related to the floral biology of walnut and how the collected samples fit into the development of the male and female flowers.  I highly suggest including larger photos of the male and female flowers representative of the samples collected.  Alternatively, there could be a detailed description of what the flowers look like at each collected date.  Which floral features are present at each collection date?  When do the flowers open?  When is the pollen mature?  When does pollination occur?  When does the fruit start to form? 

 

Another suggestion is to divide up the data presented in figure 7 into male and female flowers, or to at least use different colors or shades of grey to distinguish the two floral types.  Once the authors have clarified what floral events are occurring at each sample collection time in the male and female flowers then they can make better connections between the floral development and the gene expression data.  For example, the April 10^th sample has a very different expression pattern from the other samples.  If something is biologically different at this date (such as pollen shedding or floral opening) then it may give a better insight into the potential role of the F-box family in walnut flower.  This date also appears to be a single collection. 

 

The importance of the F-box gene family in walnut floral tissues needs to be explored in more detail.  Stating that “suggesting that the F-box gene family of common walnut was the vital genes regulating the flowering” is not a valid conclusion based on the data.  The data show that several F-box genes are enriched in floral tissues as compared to vegetative tissues.  Other analyses that could be performed would be to compare these 14 genes to each other, and to other F-box genes (do they share features or domains?).  I would also highly recommend confirming the expression of a subset of the genes and samples by QPCR. 

Author Response

Response to Reviewer 1 Comments

 

Thank you for your valuable comments. We have studied the valuable comments from you carefully, made a significant effort to make the work clearer, and tried our best to revise the manuscript. The point to point responds to the reviewer’s comments as following:

 

Comments and Suggestions for Authors

Thank you for the numerous revisions to your manuscript.  I have a few suggestions to streamline the paper and highlight the link between the bioinformatics and the biology. 

Point 1: The introduction cites a very large number of works regarding F-box genes in plants.  This section could be trimmed to better focus on the research most related to this manuscript. 

Response: Thank you for your comments. We also revised some sentences in the INTRODUCTION section from Line 36 to 41 as followings:

“The ubiquitin–proteasome pathway, which is involved in F-box protein, is one of the most important biological regulatory systems. The first F-box gene (Cyclin F) in humans was found by Kumar and Paietta, and it was called F-box [1]. F-box proteins are a component of complexes which are a substrate-recognition subunit of Skp1-Cullin1-F-box (SCF) ubiquitin–ligase. [2-4]” to “ The ubiquitin–proteasome pathway, which was involved in F-box protein, was one of the most important biological regulatory systems, F-box protein were also as a component of complexes which were a substrate-recognition subunit of Skp1-Cullin1-F-box (SCF) ubiquitin–ligase [1-4]”

We also revised some sentences in the INTRODUCTION section from Line 43 to 48 as followings:

“The protein containing the WD repeat at the C-terminus is called FBXW; FBXO is the protein containing other secondary structures at the C-terminus, such as bright ammonia and proteins such as Leucine zipper, Tetratricopeptide repeats (TPRs), Kelch, ring finger structures, and zinc finger structures [2]. Except these sequences, the F-box protein has essentially no conserved sequences [8] to “the F-box protein contained the WD repeat, Leucine zipper, Tetratricopeptide repeats (TPRs), Kelch, ring finger structures, and zinc finger structures [2,8].”

Point 2: The sample collection was better described but could use additional clarification.  Do walnut trees show synchronized floral opening such that flowers collected on very close days represent different floral ages?  Were flowers collected from equivalent positions on each tree?  Were the trees of similar ages?  Do the large clusters of flowers (as seen in the male) have synchronized development in each cluster? 

 Response: Thank you for your comments. The walnut tree which we collected was grown for around 15 years old. The flower development period of the walnut tree is actually similar, so we collected a very dense period and collected a large number of female flowers, and carried out structural anatomy, we chose the female flowers with similar cross-sectional morphology were sequenced by transcriptome, so we collected the proper samples of different periods of April 10th, April 15th, April 22th and May 1th, which were different developmental stages that can represent the development of walnut female flowers. We collected equivalent positions of the same tree and took a several of biological replicates. Yes, we collected all tissues from the same age on one tree. The growth stage of the male flower inflorescence of the walnut is indeed different. First, we collected a lot of individuals and carried out morphological anatomy, and selected three stages with obvious growth and development. The male flowers are not yet developed, and the developing male flower, and complete propagation of fully mature pollen, Finally, these selected samples were sequenced by transcriptome.

Point 3: One area that must be improved related to the floral biology of walnut and how the collected samples fit into the development of the male and female flowers.  I highly suggest including larger photos of the male and female flowers representative of the samples collected.  Alternatively, there could be a detailed description of what the flowers look like at each collected date.  Which floral features are present at each collection date?  When do the flowers open?  When is the pollen mature?  When does pollination occur?  When does the fruit start to form? 

Response: Thank you for your comments. In this study, for female flowers development stages as the first open of female flower (April 10), full date of female flower (April 15 and April 22), specifically, on April 15, the stigma is not fully developed, on April 22, the stigma is fully developed, and on May 1st is end date of female flower. For male flowers, three stages as the first open and full date of female flower (April 10 and April 11), and end date of female flower (May 2), respectively (Table S1). In this study, we did not observe the pollen developing stages (such as the slice). We searched the article from scholar database, but it was no reports on the pollen development of Juglans regia. However, we found that the common walnut August 20 to September 26 from a master’s degree thesis of Shandong Agriculture University in China. For each female flower development time, we collected female inflorescence as 3, 3, 2, and 1 biological replicate from an adult tree growing on the Qinling Mountains on April 10, April 15, April 22, and May 1, respectively (Table S1). For each male flower development time, we collected one male inflorescence from an adult tree (around 15 years old) growing on the Qinling Mountains on April 10, April 11, and May 2, respectively (for details on flower tissues collection, see Table S1).  We also added the describes in the Materials and Methods in the revision as followings: “Each tissue was collected from equivalent positions on the same tree.” and “In this study, for female flowers development stages as the first open of female flower (April 10), full date of female flower (April 15 and April 22), specifically, on April 15, the stigma is not fully developed, on April 22, the stigma is fully developed, and on May 1 is end date of female flower. For male flowers, three stages as the first open and full date of female flower (April 10 and April 11), and end date of female flower (May 2), respectively (Table S1).”, respectively.

Point 4: Another suggestion is to divide up the data presented in figure 7 into male and female flowers, or to at least use different colors or shades of grey to distinguish the two floral types.  Once the authors have clarified what floral events are occurring at each sample collection time in the male and female flowers then they can make better connections between the floral development and the gene expression data.  For example, the April 10^th sample has a very different expression pattern from the other samples.  If something is biologically different at this date (such as pollen shedding or floral opening) then it may give a better insight into the potential role of the F-box family in walnut flower.  This date also appears to be a single collection. 

 Response: Thank you for your comments. We changed the color of Figure 7 in the revision. And we revised the legends for Figure7 from Line 394 to 395 as followings: “The blue represented the expression level in female flower, the grey represented the expression level in male flower.”

For the describe of expressions, we also added some sentences in the RESULTS section from Line 377 to 381 as followings: “For male flower, we found that two genes (Jure_07156.t1, Jure_26563.t1) had higher expressed level in April 10 than other stages. For female flower, some gens (Jure_06725.t1, Jure 07950.t1, Jure_19792.t1, Jure_11456.t1, Jure_07788.t1, Jure_21178.t1, Jure_02219.t1, Jure_06252.t1, Jure_13358.t1, Jure_21675.t1, Jure_16679.t1,and Jure_05249.t1.) were expressed highly in April 10 (Figure 7).” We also added some sentences in the DISCUSSION section from Line 540 to 544 as followings: “We found that a total of 12 genes were highly expressed in  the first open of female flower (April 10), while the two genes were highly expressed in the first open of male flower, suggesting that the F-box gens were relevant in female and male flowering in common walnut (Figure 7).”

Point 5: The importance of the F-box gene family in walnut floral tissues needs to be explored in more detail.  Stating that “suggesting that the F-box gene family of common walnut was the vital genes regulating the flowering” is not a valid conclusion based on the data.  The data show that several F-box genes are enriched in floral tissues as compared to vegetative tissues.  Other analyses that could be performed would be to compare these 14 genes to each other, and to other F-box genes (do they share features or domains?).  I would also highly recommend confirming the expression of a subset of the genes and samples by QPCR. 

Response: Thank you for your comments. Yes, you are right the RT-qPCR is important for our validate the RNA expression of F-box genes. It is really hard to get the exactly same condition tissue samples from woody plants. We collected the female flowers and male flowers from different developmental stages for hardwood tree species common walnut (Juglans regia) (Table S1), and put them in liquid nitrogen as soon as possible. We performed transcriptome sequencing on high throughput sequencing platform in 2015. After that, we also saved those sample RNAs in -80 refrigerators. The expression was also analyzed by using no reference genome method. Later, the (Juglans regia) genome was reported by Martínez-García et al. 2016. Then we performed RNA-seq expression analysis based on the reference genome sequence data. As a result, the RNA samples collected in 2015 were severely degraded, so we did not try to perform RT-PCR experiment. In addition, we found that RNA expression using reference genomic analysis showed duplicate samples at different developmental stages in male and female flowers, and there were no significant differences between developmental stages. In the follow-up study, we will collect RNA samples from leaves, stems, flowers, buds and other tissues of common walnut in different growth ecosystems according to the different expression patterns in Figure 4. Concentrated on we found several potential flowering to walnuts (F-box Gene) related samples were verified by RT-PCR.

Thank you for your consideration our manuscript of “Genome-wide identification and transcriptional expression profiles of the F-box gene family in common walnut (Juglans regia L.)" publish on Journal of Forests.

 

 

Sincerely,

 

Zhao Peng, Ph.D

Author Response File: Author Response.docx

Reviewer 2 Report

Thank you for considering all my comments and editing of the language.

I have no further concerns.

Author Response

Response to Reviewer 2 Comments

 

Thank you for considering all my comments and editing of the language.

I have no further concerns.

Thank you for your review and warm comments for our manuscript. Thank you for your consideration our manuscript of “Genome-wide identification and transcriptional expression profiles of the F-box gene family in common walnut (Juglans regia L.)" publish on Journal of Forests.

 

 

Sincerely,

 

Zhao Peng, Ph.D

 

Author Response File: Author Response.doc