rolB Promotes Adventitious Root Development in Pyrus betulaefolia by Modulating Endogenous Hormones and Gene Expression

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, Wang et al. demonstrated that the rolB gene from Agrobacterium rhizogenes significantly promotes adventitious root formation and development in Pyrus betulaefolia (‘duli’) by modulating endogenous hormone levels and gene expression. Transgenic ‘duli’ plants expressing rolB exhibit earlier root primordia formation, higher rooting rates, and enhanced development of secondary roots compared to wild-type plants. The rolB gene increases the endogenous levels of auxin (IAA), gibberellic acid (GA3), and zeatin riboside (ZR), while decreasing abscisic acid (ABA) content, thereby positively influencing hormone biosynthesis, metabolism, and signaling pathways related to root growth. Transcriptomic and co-expression network analyses identified key differentially expressed genes associated with these hormone pathways and secondary metabolism, revealing underlying molecular mechanisms by which rolB controls adventitious rooting. The work is interesting however, I have some suggestions:
1.    YUC genes are key auxin biosynthesis enzymes upregulated in transgenic plants. Were IAA biosynthesis rates measured directly, or is there biochemical evidence that increased YUC expression leads to elevated auxin concentrations at the root initiation sites?
2.    The work reports hormone levels (IAA, GA3, ZR, ABA) at several time points during rooting. It would improve clarity to include detailed temporal profiles or graphs showing dynamic changes of each hormone in transgenic versus wild-type plants. 
3.    My suggestion, if possible at this stage, transcriptomic analysis identifies many differentially expressed genes (DEGs) related to hormone biosynthesis and signaling. Targeted functional validation (e.g., knockdown, overexpression, or CRISPR editing) of a subset of these key DEGs would strengthen causal links between gene expression changes and root development phenotypes.
4.    The study shows lower ABA levels in transgenic plants and downregulation of ABA biosynthesis and signaling genes, but ABA is known to inhibit rooting. Can exogenous ABA application rescue the rooting phenotype in rolB-transformed plants and thereby confirm ABA’s role?
5.    Besides hormone-related genes, are there specific transcription factors or regulatory modules directly targeted by rolB that orchestrate gene expression changes during adventitious root formation?
6.    Please strengthen the intro to rationale by clearly defining the knowledge gap and the specific objectives of the study.
7.    All figure legends are very short; please add more details about the figure to make it easily understandable for the readers. 

 

Author Response

Comments 1 :YUC genes are key auxin biosynthesis enzymes upregulated in transgenic plants. Were IAA biosynthesis rates measured directly, or is there biochemical evidence that increased YUC expression leads to elevated auxin concentrations at the root initiation sites?

Response 1 : We sincerely appreciate your valuable comment and fully accept your suggestion. This point has also been reported in previous studies, and we have accordingly added the relevant references in the manuscript to further support this statement. The detailed modifications are as follows (lines 433-436): Overall, the rolB gene increases auxin levels by up-regulating the expression of genes in the YUC biosynthesis pathway (YUC3,4,6,10) and regulating genes in the IAA signal transduction pathway (AUX12,28, IAA8,16), which in turn promotes adventitious root growth[24].

Lee, M.; Jung, J.H.; Han, D.Y.; Seo, P.J.; Park, W.J.; Park, C.M. Activation of a flavin monooxygenase gene YUCCA7 enhances drought resistance in Arabidopsis. PLANTA,2012, 235, 923-938.

Comments 2 :The work reports hormone levels (IAA, GA3, ZR, ABA) at several time points during rooting. It would improve clarity to include detailed temporal profiles or graphs showing dynamic changes of each hormone in transgenic versus wild-type plants.

Response 2 : We sincerely appreciate the reviewer’s valuable feedback. We fully agree that presenting the dynamic changes of hormone levels (IAA, GA₃, ZR, ABA) at different time points during the rooting process is crucial for a deeper understanding of the differences between transgenic and wild-type plants. In response to the reviewer’s suggestion, we have supplemented the manuscript with hormone level data at day 7 of rooting and included the corresponding dynamic profiles (line 131). We believe these additions will enhance the clarity and comprehensibility of the manuscript.

Comments 3 : My suggestion, if possible at this stage, transcriptomic analysis identifies many differentially expressed genes (DEGs) related to hormone biosynthesis and signaling. Targeted functional validation (e.g., knockdown, overexpression, or CRISPR editing) of a subset of these key DEGs would strengthen causal links between gene expression changes and root development phenotypes.

Response 3 : We sincerely appreciate your valuable comment and fully accept your suggestion. The main objective of our study is to identify genes involved in hormone pathway regulation in Pyrus betulifolia through transcriptomic analysis. We also recognize that functional validation of these genes is essential to better establish the link between gene expression and root phenotypes. Therefore, we will further conduct functional validation experiments in our future studies.

Comments 4 : The study shows lower ABA levels in transgenic plants and downregulation of ABA biosynthesis and signaling genes, but ABA is known to inhibit rooting. Can exogenous ABA application rescue the rooting phenotype in rolB-transformed plants and thereby confirm ABA’s role?

Response 4 : Thank you very much for your valuable comment. This is indeed an important point for ensuring the completeness of our study and for a deeper understanding of the role of ABA in root formation. We fully agree with your suggestion. In our future studies, we will perform exogenous ABA application experiments and examine the rooting phenotype of rolB transgenic plants to further confirm the role of ABA.

Comments 5 : Besides hormone-related genes, are there specific transcription factors or regulatory modules directly targeted by rolB that orchestrate gene expression changes during adventitious root formation?

Response 5 : Thank you for pointing this out. Transcription factors play an important role in the formation of adventitious roots. However, there are no relevant statistics or analyses of transcription factors on the cloud platform we are using. Thank you for pointing this out again. Transcription factors play an important role in the formation of adventitious roots, but there are no relevant statistics or analyses of transcription factors on the cloud platform we are using.

Comments 6 : Please strengthen the intro to rationale by clearly defining the knowledge gap and the specific objectives of the study.

Response 6 : We sincerely thank the reviewer for the valuable suggestion. We completely agree that clearly defining the knowledge gap and specific research objectives is crucial for strengthening the introduction and rationale of the study. In response to the reviewer’s comment, we have revised the manuscript to more clearly define the existing knowledge gap and the specific objectives of our study. The detailed modifications are as follows (lines: 40-54): This phenomenon has been documented in multiple plant species, highlighting the important role of rolB in root formation. Importantly, many woody plants, such as pear (Pyrus spp.), are notoriously difficult to propagate using conventional vegetative methods (e.g., cuttings and layering), which underscores the practical importance of studying rolB-mediated root induction.However, there is still a gap in understanding the specific molecular mechanisms of rolB in pear root formation, especially regarding hormone regulation. The expression of rolB in transgenic plants has been extensively documented and is associated with enlarged root systems, altered leaf and flower morphology, increased adventitious root formation, and reduced internode length. These phenotypic alterations have been consistently observed across various species transformed with rolB [2]. Although these results have been observed across species, the specific role of rolB in pear trees, particularly in regulating endogenous hormone pathways, remains unclear. This study will focus on exploring how the rolB gene affects endogenous hormone changes during adventitious root formation in pear, particularly its effect on IAA, GA3, ZR, ABA, and how these hormones interact.

Comments 7: All figure legends are very short; please add more details about the figure to make it easily understandable for the readers. 

Response 7: We sincerely appreciate your valuable suggestion. We fully agree that comprehensive and detailed figure legends are essential for helping readers clearly understand the information presented. In response to your comment, we have revised the manuscript by adding more detailed descriptions to all figure legends to improve clarity and readability. The detailed modifications are as follows: Figure 4. Analysis of endogenous hormone content of rolB-Transformed ‘duli’. (a) The content of ABA; (b) The content of IAA; (c) The content of GA3; (d) The content of ZR; (e) The content of IP. (lines: 132-133). Figure 5 . Analysis of differentially expressed genes (DEGs). (a) Distribution of DEG counts in each comparison group.(b) Venn diagram showing shared and unique DEGs during adventitious root development between rolB-transformed and wild-type Pyrus betulaefolia at different time points. Comparison groups in-clude DBCK vs DCK, DB1 vs D1, DB5 vs D5, DB10 vs D10, and DB20 vs D20. (lines: 143-146). Figure 7. KEGG enrichment analysis of DEGs between rolB-transformed and WT ‘duli’ during adventitious root development. (a) KEGG enrichment at 0 days (DB0 vs D0);(b) KEGG enrichment at 1 day (DB1 vs D1);(c) KEGG enrichment at 5 days (DB5 vs D5);(d) KEGG enrichment at 10 days (DB10 vs D10);(e) KEGG enrichment at 20 days (DB20 vs D20). (lines: 173-176). Figure 8. Clustering and correlation analysis of hormone and modular genes. (a) Hier-archical clustering analysis of co-expression genes. Different colors represent all mod-ules, with gray indicating genes that cannot be classified into any module by default. (b) Correlated heat maps between modules. A color block in the picture represents a nu-merical value. The redder the color, the higher the expression level, and the bluer the color, the lower the expression level. (c) Correlations between gene modules and phe-notypes. Each tree diagram in the figure represents a module, each branch represents a gene, and the darker the color of each point (white → yellow → red), the stronger the connectivity between the two genes corresponding to the row and column. (d) Heat map of correlations between gene modules and hormone. The leftmost color block represents the module, and the rightmost color bar represents the correlation range. In the heatmap of the middle part, the darker the color, the higher the correlation, with red indicating positive correlation and blue indicating negative correlation. The num-bers in each cell represent correlation and significance. (lines: 187-200). Figure 9. (a) DEGs expression pattern in pink module.Above is the expression calorimetric map of DEGs in different samples, and the abscissa is the sample name. In the figure below, the horizontal coordinate is the sample name, and the vertical coordinate is the expression quantity.(b) GO en-richment analysis of hub genes in pink module; (c) KEGG enrichment analysis of key genes in pink module;(d) Co-expression network of hub genes in pink module. the text inside the node was the gene name, and the node size and color depth were the kWithin value of the gene, which repre-sented the connectivity of the gene within the module. (e) Heatmap illustrating the expression patterns of genes associated with auxin biosynthesis, metabolism, and signaling pathways. Red indicates upregulation, while blue indicates downregulation of gene expression. (lines: 231-239)

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

   I was pleased to be selected as a reviewer for the manuscript entitled “rolB Promotes Adventitious Root Development in Pyrus betulaefolia by Modulating Endogenous Hormones and Gene Expression”. The aim of the study was to examine the effect of the transformation of the rolB gene on root development and hormone concentration in Pyrus betulaefolia and thus, it fits the aims and scope of the journal. The manuscript provided sufficient background and a detailed explanation of why it was important to study the rolB gene and why the research was done. The results are significant and written clearly, as so the methods section. Some figures need to be improved. Discussion provides a detailed explanation of the mechanism and role of particular genes in relation to rolB gene. The manuscript requires some corrections, but there are no significant flaws; thus, I recommend a minor revision.

I have a few comments for the Authors:

1. Line 11 – Beginning of the abstract is missing.
2. In the introduction section, there is no direct information about the aim of this study. Please consider adding it.
3. Figure 2 – Is it possible to enlarge the description of the colour bars?
4. There are no direct references to particular parts of figures in the text (e.g. Figure 3A, Figure 3B, etc.). Please add them and check for all figures.
5. Figure 3 – I cannot see which colour stands for WT and which for transformed plants in the figure. There is only “D” and “DB”, which are not explained. Please add this information. Similarly, check this in Figure 4. Also, in lines 128-130, please explain in the result section the abbreviations “D”, “DB”, etc., to make reading easier.
6. Chapter 2.3. Please adjust the text so that the description of particular hormones is in the same order as they are presented in Figure 4.
7. Figures 6, 7, 8, 9, 10, 11, 12, 13, 14 - The descriptions in the figures are not visible; please enlarge them.
8. Figure 9 – please add description and the next letter to the last chart. Similar to figures 10, 11, 12.
9. Line 299 – Table X should be numbered.
10. Line 343, 495, 543 – Please add the year of the cited articles.
11. Line 374, 388, 402 – In my opinion, abbreviations should be explained. Please consider adding abbreviations list.

Author Response

Comments 1 : Line 11 – Beginning of the abstract is missing.

Response 1 : Thank you for pointing out the missing introduction at the beginning of the abstract. I appreciate your suggestion. We have now added the introductory sentence to the abstract as suggested. The results of the revisions are as follows (lines 11-13): We investigated the effect of Agrobacterium rhizogenes-mediated transformation mof rolB on adventitious root development and endogenous hormones in ‘duli’ (Pyrus betulaefolia) via transcriptomic analysis of wild-type (WT) and rolB-transformed plants.

Comments 2 : In the introduction section, there is no direct information about the aim of this study. Please consider adding it.

Response 2 : Thank you for your valuable suggestion. I appreciate your suggestion. We have now added the direct information regarding the aim of the study in the introduction section. The results of the revisions are as follows (lines 67-69): Thus, this study aims to specifically explore how rolB regulates hormone synthesis, me-tabolism, and signaling pathways during root initiation, filling a critical gap in current research.

Comments 3 : Figure 2 – Is it possible to enlarge the description of the colour bars?

Response 3 : Thank you for your valuable suggestion regarding the description of the color bars in Figure 2. I appreciate your suggestion. We agree that improving the readability of the color bars is important. In response, we have modified Figure 2 by enlarging the description of the color bars to make it easier for viewers to interpret (lines 102-104).

Comments 4 : There are no direct references to particular parts of figures in the text (e.g. Figure 3A, Figure 3B, etc.). Please add them and check for all figures.

Response 4 : Thank you for raising this important point. We agree that directly referring to specific parts of the figures in the text can significantly improve readability and clarity. In response to your suggestion, we have revised the manuscript by adding references to the relevant parts of all figures to facilitate readers’ understanding and interpretation (lines 121-125).

Comments 5 : Figure 3 – I cannot see which colour stands for WT and which for transformed plants in the figure. There is only “D” and “DB”, which are not explained. Please add this information. Similarly, check this in Figure 4. Also, in lines 128-130, please explain in the result section the abbreviations “D”, “DB”, etc., to make reading easier.

Response 5 : Thank you for pointing out these issues. I completely understand the importance of clarifying the abbreviations and ensuring that the figure legends are easy to interpret. I appreciate your suggestion. I have added explanations for the abbreviations "D", "DB", etc., in both Figure 3 and Figure 4, and I also clarified which colors represent WT and transformed plants. The results of the revisions are as follows (lines 107-108): After 30 days of rooting treatment, the rooting rate was 98.01% for rolB-transformed ‘duli’(D)and 79.14% for WT plants(DB) (Figure 3).

Comments 6 : Chapter 2.3. Please adjust the text so that the description of particular hormones is in the same order as they are presented in Figure 4.

Response 6: Thank you for your valuable feedback. I fully accept the reviewer’s suggestions and have made the necessary adjustments. The order of the hormone descriptions in Chapter 2.3 has been modified to match the sequence in Figure 4. The results of the revisions are as follows (lines 119-130): The rolB gene significantly increased the content of endogenous IAA, GA3, and ZR during the formation of adventitious roots and decreased the content of endogenous ABA (Figure 4a). The endogenous IAA content of transgenic (DB) and WT ‘duli’(D) did not significantly differ, and the endogenous IAA content of transgenic ‘duli’ and WT ‘duli’ was 56.89% and 44.42% lower at 20 days of rooting treatment(Figure 4b), respectively, than at 0 days. No significant changes in the endogenous GA3 and ZR content of WT ‘duli’ within 20 days of rooting were observed(Figure 4c) (Figure 4d); no significant changes were observed in the endogenous GA3 and ZR of transgenic ‘duli’ within 10 days of rooting, but a rapid decrease in the content of GA3 and ZR was observed after 10 days. The endogenous ABA content in transgenic ‘duli’ and WT ‘duli’ did not change signifi-cantly within 10 days of rooting, but decreased significantly after 10 days. Differences in the endogenous IP(Isoamyl alkenyl adenine) content of transgenic and WT ‘duli’ were observed(Figure 4e).

Comments 7 : Figures 6, 7, 8, 9, 10, 11, 12, 13, 14 - The descriptions in the figures are not visible; please enlarge them.

Response 7 : Thank you for your valuable feedback. I fully understand the importance of ensuring that figure descriptions are clearly visible to aid in the readers' understanding. I have enlarged the descriptions in Figures 6, 7, 8, 9, 10, 11, 12, 13, and 14 to enhance clarity and make them easier to read. Your suggestion has greatly improved the accessibility of these figures (lines 158, 173, 187, 230, 252, 327, 339, 355, 357).

Comments 8 : Figure 9 – please add description and the next letter to the last chart. Similar to figures 10, 11, 12.

Response 8 : Thank you for your valuable suggestion. I fully understand the importance of adding descriptions and letters to ensure clarity and improve the reader’s understanding. I have added the necessary letters and descriptions to the last chart in Figure 9 ( line 231), in a manner similar to Figures 10, 11, and 12. This should make the figures more accessible and easier to interpret.

Comments 9 : Line 299 – Table X should be numbered.

Response 9 : Thank you for highlighting this point. I completely understand the importance of proper table numbering to maintain clarity and consistency throughout the manuscript. I have added Table 1 and corrected the numbering accordingly (line354). This adjustment ensures that the references in the text and the tables are aligned and easy to follow.

Comments 10 : Line 343, 495, 543 – Please add the year of the cited articles.

Response 10 : Thank you for your helpful suggestion. I have made the necessary revisions and added the years of the cited articles in lines 343, 495, and 543 as requested. This should improve the accuracy and clarity of the references (lines 398, 551, 599).

Comments 11 : Line 374, 388, 402 – In my opinion, abbreviations should be explained. Please consider adding abbreviations list.

Response 11 : Thank you for your helpful suggestion. I have accepted your comment and made the corresponding revisions in the manuscript, explaining the meaning of each abbreviation to ensure clarity for the readers (lines 416-417, 427, 440-449). The results of the revisions are as follows (lines 11-13): Plant hormones such as gibberellins(GA), abscisic acid(ABA), brassinolide(BR), and strigolactones(SLs) are also derived from terpenoids. the Aux/IAA proteins interact with TOPLESS proteins to inhibit the activity of specific ARFs (auxin respond factors). The first stage occurs in plastids, where the GA precursor GGPP(Geranylgeranyl pyro-phosphate) is cyclized into ent-kaurene by the enzymes CPS(Copalyl pyrophosphate synthase) and KS (Endogen-shell synthase). The second stage occurs in the endoplasmic reticulum, where KO (Endogen-kauri oxidase) and KAO (Endogen-kauri acid oxidase) catalyze the oxidation of ent-kaurene, forming the initial GA product GA12-aldehyde, which is further converted into GA12 and GA53. The third stage occurs in the cytoplasm, where GA12 and GA53 are converted into other forms of GAs through oxidation by enzymes such as GA20ox(GA20-oxidase), GA3ox(GA30-oxidase), and GA2ox [25–27]. Thus, the rolB gene influences GA biosynthesis in the endoplasmic reticulum.

 

Author Response File: Author Response.pdf