A Genome-Wide Characterization of the Xyloglucan Endotransglucosylase/Hydrolase Family Genes and Their Functions in the Shell Formation of Pecan
and Anmin Yu 1,3,4,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis manuscript presents a genome-wide analysis of the XTH gene family in Carya illinoinensis, focusing on their role in shell formation. The study is well-structured and offers valuable insights into cell wall remodeling genes and their potential influence on nut shell development. The integration of phylogenetic, structural, and expression data is comprehensive and informative. I recommend acceptance after minor revisions.
The introduction would benefit from a clearly defined research hypothesis at the end. While the background is well explained, it remains unclear what the authors specifically set out to test or demonstrate.
Line 139: It is not clear why both BLASTp and HMMER searches were used. Since HMMER is generally sufficient for domain-based searches, a rationale for including BLASTp should be provided. Additionally, clarify whether candidate genes were required to contain both domains (Glyco_hydro_16 and XET_C) or if one was sufficient.
Figure 1A: Please improve the color scheme for better visibility. The font size in the gene structure and domain diagrams should also be increased.
Figure 5A and 5B: Font size is too small for comfortable reading. Please enlarge the labels in both panels to improve clarity.
Author Response
Comment 1: Line 139: It is not clear why both BLASTp and HMMER searches were used. Since HMMER is generally sufficient for domain-based searches, a rationale for including BLASTp should be provided. Additionally, clarify whether candidate genes were required to contain both domains (Glyco_hydro_16 and XET_C) or if one was sufficient.
Response 1: Thank you for this insightful comment. We employed both BLASTp and HMMER in our gene‐family identification for the following reasons: BLASTp is fast and effective for detecting highly conserved homologs based on full‐length protein‐sequence similarity (Xu et al. 2019; Zhu et al. 2014). HMMER, using the Pfam HMM profiles for Glyco_hydro_16 (PF00722) and XET_C (PF06955), enables a genome‐wide scan that not only captures the highly conserved family members but also identifies more divergent proteins that still retain key catalytic or binding domains (Yokoyama & Nishitani 2001). By combining these two approaches, we maximize coverage of the canonical XTH homologs while maintaining sensitivity to detect remotely related members, thereby minimizing both false negatives and false positives.
Regarding domain criteria in our HMMER search: we treated Glyco_hydro_16 and XET_C as independent HMM queries. Any protein containing either domain was retained as an XTH candidate, and subsequent multiple‐sequence alignment and phylogenetic analysis were used to confirm its placement within the XTH family. This strategy follows the methodological precedent set by Xu et al. (2019) and Zhu et al. (2014).
Reference:
Xu et al. (2019) “Genome-wide identification and expression profiling of the xyloglucan endotransglucosylase/hydrolase (XTH) family in tomato” Plant Physiology and Biochemistry 138: 231–242.
Zhu et al. (2014) “Genome-wide identification, phylogeny, and expression analysis of the XTH gene family in Populus trichocarpa” Tree Genetics & Genomes 10: 1251–1263.
Yokoyama & Nishitani (2001) “A comprehensive expression analysis of all members of a gene family encoding cell-wall enzymes revealed distinct but overlapping expression patterns for two poplar XTH genes” Plant Physiology 127(3): 998–1006.
Comment 2: Figure 1A: Please improve the color scheme for better visibility. The font size in the gene structure and domain diagrams should also be increased.
Response 2: Thank you for your helpful suggestion. We have revised Figure 1A (now renamed as Figure 2) by improving the color scheme to enhance visual contrast and readability. In addition, the font size in both the gene structure and protein domain diagrams has been increased to ensure better clarity and visibility.
Comment 3: Figure 5A and 5B: Font size is too small for comfortable reading. Please enlarge the labels in both panels to improve clarity.
Response 3: We appreciate the reviewer’s comment. The font size in both Figure 5A and Figure 5B (now renamed as Figure 7A and Figure 7B) has been enlarged to improve legibility. The updated figures now present the labels more clearly to facilitate comfortable reading.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors studied the genome-wide characterization of the XTH gene family in pecan and identified 38 XTH genes, which were categorized into four distinct phylogenetic groups. These genes were bioinformatically characterized, and their expression was evaluated using RNA-seq database. Overall, the article is well written. However, there is information that seems to be missing in the methodology.
MAYOR CHANGES
Lines 135-136. The authors described the removal of the green exocarp and
seed tissues, leaving only the shell tissues, which were immediately frozen in liquid nitrogen and stored at -80℃ for further analysis. However, there is no description of RNA extraction or other analyses for these samples. Is this missed methodology related to the results in Figure 4B?
I can't find Figure S1 in the supplementary archives mentioned in section 2.1.
Figure 4. Include the acronyms KD1, 2, 3, and SX1, 2, 3 in the legend. What is the methodology description for these samples?
MINOR CHANGES
Line 24: I suggest Structural analyses of the deduced proteins
Line 28, "formaiton" should be "formation".
Line 96, "were" should be "and it is".
Line 135, "immediatly" should be "immediately".
Author Response
Comment 1: Lines 135-136. The authors described the removal of the green exocarp and seed tissues, leaving only the shell tissues, which were immediately frozen in liquid nitrogen and stored at -80℃ for further analysis. However, there is no description of RNA extraction or other analyses for these samples. Is this missed methodology related to the results in Figure 4B?
Response 1: We thank the reviewer for pointing this out. The missing methodology has now been added in section 2.6 (Lines 184–199) to describe the RNA extraction, library construction, and RNA-seq analysis performed on the shell tissue samples, which are indeed related to the results presented in Figure 4B (now renamed as Figure 6B).
Comment 2: I can't find Figure S1 in the supplementary archives mentioned in section 2.1.
Response 2: We apologize for the oversight. Figure S1 was unintentionally omitted during submission. It has now been included in the revised supplementary materials.
Comment 3: Figure 4. Include the acronyms KD1, 2, 3, and SX1, 2, 3 in the legend. What is the methodology description for these samples?
Response 3: Thank you for the suggestion. We have updated the Figure 4 legend to include the sample acronyms KD1, KD2, KD3, SX1, SX2, and SX3 for clarity. Additionally, the methodology for sample collection and RNA-seq analysis has been described in detail in the newly added section 2.6 (Lines 184–199).
MINOR CHANGES
Comment 4: Line 24: I suggest Structural analyses of the deduced proteins
Response 4: Revised as suggested (Line 25).
Comment 5: Line 28, "formaiton" should be "formation".
Response 5: Corrected. The typo “formaiton” has been changed to “formation” (Line 29).
Comment 6: Line 96, "were" should be "and it is".
Response 6: Revised accordingly. The phrase has been changed from “were” to “and it is” to improve grammatical accuracy (Line 98).
Comment 7: Line 135, "immediatly" should be "immediately".
Response 7: Thank you for pointing this out. The spelling error has been corrected to “immediately”.
Reviewer 3 Report
Comments and Suggestions for AuthorsIn this study authors presents a comprehensive genome-wide analysis of the XTH gene family in pecan, identifying 38 genes classified into four phylogenetic groups.
The work highlights conserved and divergent structural features suggesting functional specialization.
Expression analyses across tissues and shell phenotypes implicate specific XTHs in lignified shell development.
The findings offer valuable insights into shell formation and provide a foundation for genetic improvement in pecan and related species. To improve the content of the manuscript please find the following comments.
- Line 55to 59 the sentence are repetitive make it one sentence.
- Figure 1c, show the confidence of the model that is pLDDT score, with color-coded model and the figures not clear, please show the differences in the loop in inset.
- Line 201, please correct the figure number from “1A” to “1B”. 1A is about phylogenetic analysis and not about ExDxE motif.
- Line 206-07, not correlation between mentioned base pairs and the aa range. Please correct it.
- Line 208, authors write the protein are basic in nature. Please mention the percentage of the basic amino acids present in the protein ( may be in range) or add one more column in Table 1.
- Line 215, Keep the same nomenclature as figure 1B.
- Make Figure S3 as main figure.
- Instead of this annotation 116 (G), 117 (F), please change it to G116 and F117.
- The conclusion that group I and II genes are ancestral (line 313) is not well supported by the presented evidence. Higher Ks values may indicate older duplication events, but this should be explicitly discussed with supporting references or broader context.
- It is unclear how "purifying selection" is inferred beyond the Ka/Ks < 1 threshold. A brief explanation of how this result aligns with functional constraints within the gene family would enhance interpretation.
Author Response
Comment 1: Line 55 to 59 the sentence are repetitive make it one sentence.
Response 1: Thank you for your suggestion. We have revised the above sentences o eliminate redundancy and combined them into a single, concise sentence for improved clarity and flow (Line 55 to 59).
Comment 2: Figure 1c, show the confidence of the model that is pLDDT score, with color-coded model and the figures not clear, please show the differences in the loop in inset.
Response 2: We appreciate this helpful suggestion. We have now added a color-coded pLDDT confidence score and add the Figure S4 to indicate the model's reliability. Additionally, we have included an inset to clearly highlight the differences in the loop regions in Figure 3, as requested. The updated figure improves both clarity and interpretability.
Comment 3: Line 201, please correct the figure number from “1A” to “1B”. 1A is about phylogenetic analysis and not about ExDxE motif.
Response 3: Thank you for pointing this out. We have corrected the figure number in line 201 from “1A” to “1B” (now renamed as Figure 1) accordingly.
Comment 4: Line 206-07, not correlation between mentioned base pairs and the aa range. Please correct it.
Response 4: Thank you for your careful observation. We have revised the sentence to accurately reflect the relationship between the base pairs and the corresponding amino acid range (Line 234).
Comment 5: Line 208, authors write the protein are basic in nature. Please mention the percentage of the basic amino acids present in the protein ( may be in range) or add one more column in Table 1.
Response 5: Thank you for this valuable suggestion. We have now included the percentage range of basic amino acids in the text (line 241) and also added an additional column in Table 1 to report the proportion of basic residues in each protein.
Comment 6: Line 215, Keep the same nomenclature as figure 1B.
Response 6: Thank you for pointing this out. We have revised the text to ensure consistent nomenclature with Figure 1B (now renamed as Figure 2).
Comment 7: Make Figure S3 as main figure.
Response 7: As suggested, we have moved Figure S3 to the main figures and renumbered it as Figure 2 accordingly in the revised manuscript.
Comment 8: Instead of this annotation 116 (G), 117 (F), please change it to G116 and F117.
Response 8: We have revised the annotations as recommended. The format now appears as "G116 and F117" in the revised manuscript.
Comment 9: The conclusion that group I and II genes are ancestral (line 313) is not well supported by the presented evidence. Higher Ks values may indicate older duplication events, but this should be explicitly discussed with supporting references or broader context.
Response 9: We thank the reviewer for pointing this out. In the revised manuscript, we have added a more detailed explanation of how higher Ks values are often interpreted as indicators of older duplication events, and we now cite relevant studies (e.g., Blanc and Wolfe, 2004) to support this interpretation. We also expanded the discussion on the differences between segmental and tandem duplications to better justify the conclusion that group I and II genes are more ancestral.
Comment 10: It is unclear how "purifying selection" is inferred beyond the Ka/Ks < 1 threshold. A brief explanation of how this result aligns with functional constraints within the gene family would enhance interpretation.
Response 10: Thank you for the valuable suggestion. We have now expanded our interpretation of the Ka/Ks results by briefly explaining that Ka/Ks < 1 indicates purifying selection, which reflects the removal of deleterious non-synonymous mutations. This aligns with the known functional importance of XTH genes in cell wall modification, suggesting that these genes are under strong evolutionary constraint.
Reviewer 4 Report
Comments and Suggestions for AuthorsBasic reporting
The authors investigate the XTH gene family in pecan (Carya illinoinensis), focusing on the role of these cell wall modifying enzymes in the shell formation. The work is supporter by a RNA-seq data published previously (ref nº38). The writing is clear, unambiguous and technically correct. The structure of the article has an acceptable format for Horticulturae.
The introduction has sufficient and up-to-date background information. The figures and tables chosen by the authors are adequate to show the results of the research. The graphs are not of sufficient resolution (Figure 1 and 5) and it should be imporved. Figures are correctly described and labelled.
Experimental design
Manuscript clearly shows the research question in lines 113-119. Methods are well described. Experimental design is correct. The results are well described and correspond to the figures. The discussion is of sufficient quality, however I recommend including a more in-depth discussion on the “hotspot and crosslinking” role of xyloglucan. The conclusions are adequate, connected to the original research question and supported by the results.
Additional comments
No additional comments.
Author Response
Comment: Manuscript clearly shows the research question in lines 113-119. Methods are well described. Experimental design is correct. The results are well described and correspond to the figures. The discussion is of sufficient quality, however I recommend including a more in-depth discussion on the “hotspot and crosslinking” role of xyloglucan. The conclusions are adequate, connected to the original research question and supported by the results.
Response: Thank you for the valuable suggestion. We have now added the discussion on the “hotspot and crosslinking” role of xyloglucan (Line 490-497).
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors studied the genome-wide characterization of the XTH gene family in pecan and identified 38 XTH genes, which were categorized into four distinct phylogenetic groups. These genes were bioinformatically characterized, and their expression was evaluated using the RNA-seq database. Overall, the article is well written, and the authors made all the suggested changes.
Reviewer 3 Report
Comments and Suggestions for AuthorsNA
