Lignification and Gene Expression Pattern in Postharvest Moso Bamboo Shoots
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Editor, Dear authors,
This article concerns “Lignification and Gene Expression Pattern in Postharvest Moso Bamboo Shoots, by Xujie Huang, Yaling Zhang, Nianjun Huang, Yonglong Li, Fen Yu, Wengen Zhang and Chunce Guo. As interesting and quite rare approach I recommend it for an international audience in this journal, however several points have to be considered by the authors and a major revision is requested.
Please notice that in order to bring a broad audience to this article and to this journal, for specialists and non-specialists, the 5 major points of my comments (at the beginning) are very important (mandatory…) for a suitable value of the article. Minor points are also enhanced at the end of this review.
Sincerely yours,
The 5 major points are:
1- The main point embarrassing me a lot is that although it is a very good and rare idea to refer to anatomical sections, they are actually too weakly described (in 3.1. "showed significant morphological changes" and in 3.2. "the trends in the changes of these three contents were consistent" are not enough sustained) and not relevant, much more relevant data necessitate to quantify and evaluate very precisely the surface of xylem, the proportion of xylem in a section, the number of cells involved and their sizes, the thickness of xylem... (you can do this for instance with Imaje J or any other software); then you can proceed to statistical analyses (for instance t student tests but with a sufficient number of data; in this respect, in 2.3 the total number of samples is not clear, is it 40 or 40X10 times interval=400?) to see if there are significant differences (very relevant for your topics) or not (= just tendencies, much less relevant of course) between the anatomical sections of different dates. This is necessary to increase a lot the value of your paper.
2- 1/ since many genes are involved sensu lato in lignification however with very large (other?) functions as related in this paper (stress response, photosynthesis...) more or less far from your lignification focus, 2/ since the comparisons with pathways from other plants is quite criticized nowadays as these other plants correspond to other genomes and families, the relationship between genes and lignification is not clear and your conclusions should be much more cautious. Actually I do not see clearly the relationship between the phenotypes observed and the putative function(s) of the genes; there are (so) many correlations in plants but correlation and function are two different aspects not automatically connected; a correlation between gene and phenotype character (= lignification in your case) may exist, however the “real” function of a gene(s) is a very complex concept where “chain(s) of causation” has to demonstrate that for instance in your case lignification is caused by a given gene(s), which needs detailed physiology and anatomy and molecular (and etc…) studies, usually a very hard work!
3-3 Linked with point 1 above, 1/ for figure 2, since it is one of the bases of your article the photos are far too small, the sections are not enough contrasted and the differences between the tissues are almost not detectable; moreover put the value of the scale bar directly beside the scale, in order to be read more rapidly. 2/ for figure 3, the photos are also far too small, the contrasts are not enough enhanced and almost not visible in the sections: xylem should be much more green-blue, phloem light pink; moreover indicate that A-J are light microscopy (?); finally we do not see clearly which tissue corresponds to the (welcome) arrows. 3/ point 3.1. is not clear at all: the use of VA and PI is not understandable in terms of anatomy (refer to the terminology of types of cells and tissues as named in classical botanical anatomy; "the fresh bamboo shoots..." sentence has no verb ? "becoming largely inedible and losing their nutritional value" is an interpretation which has no anatomical support.
4- The PCA of figure 5 is far too weakly developed. As the PCA is just relating about the samples and not on the characters used for the matrix describing each sample, its interest is too weakly developed: It is usually of great interest to check about the importance of characters and etc…; if PCA is realized with supplemental data S3 (?), please detail each character contribution (line 2 of this S3) and their interest in your study. The percentage of contribution of each axis (1 and 2 I suppose?) I also very useful to state about the hierarchy between the characters and between the samples. Please check all these in mathematical tutorials easily found in internet.
5- References already taken in account by the authors are of interest, however checking briefly in web of science WOS and scilit (from mdpi) with some key-words of this manuscript, (many…) other references appear for this largely studied topics and they should be updated and used (if relevant…) in order to sustain much more and provide a larger view of these researches. Among recent papers are the followings:
[1-14]
1. Zhang, W.; Shi, M.; Yang, K.; Zhang, J.; Gao, Z.; El-Kassaby, Y.A.; Li, Q.; Cao, T.; Deng, S.; Qing, H.; et al. Regulatory networks of senescence-associated gene-transcription factors promote degradation in Moso bamboo shoots. Plant Cell Environ 2024, 47, 3654-3667, doi:10.1111/pce.14950.
2. Yang, K.; Shan, X.; Shi, J.; Zhu, C.; Gao, Z. Identification and Expression Analysis of 4CL Gene Family in Phyllostachys edulis. Journal of Nuclear Agricultural Sciences 2021, 35, 72-82.
3. Yang, K.; Li, Z.; Zhu, C.; Liu, Y.; Li, H.; Di, X.; Song, X.; Ren, H.; Gao, Z. A hierarchical ubiquitination-mediated regulatory module controls bamboo lignin biosynthesis. Plant Physiology 2024, doi:10.1093/plphys/kiae480.
4. Wang, W.; Li, Y.; Cai, C.; Zhu, Q. Auxin response factors fine-tune lignin biosynthesis in response to mechanical bending in bamboo. New Phytologist 2024, 241, 1161-1176, doi:10.1111/nph.19398.
5. Wang, K.-L.; Zhang, Y.; Zhang, H.-M.; Lin, X.-C.; Xia, R.; Song, L.; Wu, A.-M. MicroRNAs play important roles in regulating the rapid growth of the Phyllostachys edulis culm internode. New Phytologist 2021, 231, 2215-2230, doi:10.1111/nph.17542.
6. Wang, B.; Chen, S.; Zhu, Z.; Li, Y.; Li, C.; Zhang, Y.; Huang, Y.; Xu, G.; Zhao, X.; Cao, Y.; et al. PeMYB26, an R2R3-MYB transcription factor, positively regulates lignin deposition in Moso bamboo. Plant Growth Regul 2024, doi:10.1007/s10725-024-01236-9.
7. Sun, K.; Jiang, J.; Ding, Y.; Ramakrishnau, M.; Wei, Q. Morphological and anatomical analyses of moso bamboo culm necks. Journal of Nanjing Forestry University. Natural Sciences Edition 2021, 45, 40-46.
8. Que, F.; Liu, Q.; Zha, R.; Xiong, A.; Wei, Q. Genome-Wide Identification, Expansion, and Evolution Analysis of Homeobox Gene Family Reveals TALE Genes Important for Secondary Cell Wall Biosynthesis in Moso Bamboo (Phyllostachys edulis). Int J Mol Sci 2022, 23, doi:10.3390/ijms23084112.
9. Mu, C.; Jiang, J.; Fang, H.; Cheng, W.; Wu, C.; Geng, R.; Cheng, Z.; Gao, J. Unraveling developmental patterns and differentiation trajectories in a single developing internode of Moso Bamboo (Phyllostachys edulis). Ind Crop Prod 2024, 222, doi:10.1016/j.indcrop.2024.119646.
10. Li, Z.; Yuan, T.; Zhu, C.; Yang, K.; Song, X.; Gao, Z. Molecular Characteristics and Patterns of Gene Expression of Ammonium Transporter in Moso Bamboo. Scientia Silvae Sinicae 2021, 57, 70-79.
11. Li, Z.; Yang, K.; Zhu, C.; Liu, Y.; Guo, D.; Xiao, X.; Gao, Z. Identification and Expression Pattern Analysis of ABCG Genes in Moso Bamboo. Journal of Nuclear Agricultural Sciences 2023, 37, 917-926.
12. Li, Y.; Zhang, S.; Zhang, D.; Li, X.; Gao, Z.; Jiang, Z. The miR166-mRNA network regulates vascular tissue differentiation in Moso bamboo. Frontiers in Genetics 2022, 13, doi:10.3389/fgene.2022.893956.
13. Li, H.; Li, Z.; Yang, K.; Lin, Z.; Zhu, C.; Liu, Y.; Gao, Z. Comparison analysis of ABCG subfamily in bamboo and the potential function of PeABCG15 in monolignol transport. Plant Physiology and Biochemistry 2024, 217, doi:10.1016/j.plaphy.2024.109278.
14. Li, G.; Zhu, C.; Yang, K.; Wang, X.; Gao, Z. Identification and Expression Pattern Analysis of C4H Genes in Phyllostachys edulis. Journal of Tropical and Subtropical Botany 2022, 30, 151-160.
Minor points are:
1 As I am involved in plant taxonomy I am very sensible to correct plant taxa names which make their homogeneity and precision at the international level. In this respect the names of the author(s) (not in italics) have to be inserted (at least) the first time they appear in the text (from the beginning of the introduction), the whole text (especially for 2.7, see also for figure 8; see for all other parts) has to be revised carefully as there are mistakes. Use international Plant Names Index (IPNI) https://www.ipni.org/ or equivalent.
2 Since palatability is not studied, I wonder if this word is relevant in the abstract, it would be better in the introduction or in the conclusion for further studies?
3 In 2.1, indicate in botanical terms the meaning of "the third section... excluding...", do you mean the third crown of leaf bases ? Indicate in full letters the meaning of FAA.
4 For figure 1, change for "treatment", indicate for (a) the middle part and outer part of the section, (b) and (c) are not understandable in their precise location in the section.
5 In 2.2, indicate the meaning of "transparency", also the chemicals for staining. The sentence "Anatomical changes..." should be placed before "microphotographs were ..." ?
6 For figure 4, indicate if it is the standard deviation of confidence interval which is indicated for each mean.
7 For figure 5 B, indicate what means the horizontal and vertical axes; for the PCA A, indicate the meaning of each of the three sets of data.
8 Restrict results to only raw data and delete all sentences belonging actually to the discussion ("suggesting" in 3.3," indicate" in 3.4., "may be attributed" in 3.5; see for all other parts.
9 For figure 6, put all your texts in bold letters, they are almost invisible as too small; precise the meanings of the scales as the very short title used in not understandable; indicate also in full letters DEG and GO, a figure has to be understood by itself, not referring to the text.
10 For figure 7, for the same reasons indicate in full letters in B the meaning of WGCNA and DEG; indicate also the meaning of the colors; moreover B is far too small and almost invisible.
11 For figure 8, use the same format of letters for each taxon; moreover if the values correspond to former articles indicate them.
12 For figure 9, indicate in full letters the meaning of DEG.
Author Response
Dear Reviewer,
Thank you for considering our manuscript and giving us the opportunity to address your concerns and comments. We greatly appreciate your efforts, and your insightful feedback and suggestions have been invaluable in improving our manuscript. We have followed your instructions and considered the comments carefully and revised the manuscript accordingly. Our detailed responses are provided below after the specific comments, so that you can clearly see how we have revised the manuscript.
This article concerns “Lignification and Gene Expression Pattern in Postharvest Moso Bamboo Shoots, by Xujie Huang, Yaling Zhang, Nianjun Huang, Yonglong Li, Fen Yu, Wengen Zhang and Chunce Guo. As interesting and quite rare approach I recommend it for an international audience in this journal, however several points have to be considered by the authors and a major revision is requested.
Please notice that in order to bring a broad audience to this article and to this journal, for specialists and non-specialists, the 5 major points of my comments (at the beginning) are very important (mandatory…) for a suitable value of the article. Minor points are also enhanced at the end of this review.
Response: We thank the reviewer for the positive comments and for all recommendations for improving our manuscript. We have made extensive revisions to accommodate the suggestions and criticisms, with detailed descriptions of the changes presented below in response to specific comments.
The 5 major points are:
1) The main point embarrassing me a lot is that although it is a very good and rare idea to refer to anatomical sections, they are actually too weakly described (in 3.1. "showed significant morphological changes" and in 3.2. "the trends in the changes of these three contents were consistent" are not enough sustained) and not relevant, much more relevant data necessitate to quantify and evaluate very precisely the surface of xylem, the proportion of xylem in a section, the number of cells involved and their sizes, the thickness of xylem... (you can do this for instance with Imaje J or any other software); then you can proceed to statistical analyses (for instance t student tests but with a sufficient number of data; in this respect, in 2.3 the total number of samples is not clear, is it 40 or 40X10 times interval=400?) to see if there are significant differences (very relevant for your topics) or not (= just tendencies, much less relevant of course) between the anatomical sections of different dates. This is necessary to increase a lot the value of your paper.
Response: We appreciate your suggestion to provide a more detailed and quantifiable description of the anatomical sections. In response, we used ImageJ software to perform a more precise quantification of the primary xylem area, proportion, diameter, and thickness, as shown in Figure 3. The relevant data are now included in Supplementary Table S3 and Supplementary Figure S2, with statistical analyses added to support the results. This strengthens the connection between the anatomical observations and the main focus of the study.
We also revised several sections to improve the clarity and relevance of the anatomical descriptions. For example, we modified the sentence:
"The transverse sections of Moso bamboo shoots stored at different time intervals displayed varying degrees of morphological changes." (Line180-181) and removed the phrase "the trends in the changes of these three contents were consistent".
We also updated the manuscript to include more rigorous analyses, such as: “Meanwhile, we measured the changes in the area, thickness, and diameter of the primary xylem in vascular bundles over time (Supplementary Table S3). The data show that the primary xylem exhibited distinct trends with increasing storage time (Figure S2). Specifically, the area and diameter of the primary xylem decreased rapidly from 0 to 4 hours, then gradually recovered after 6 hours, showing a significant increase by 12 hours. In contrast, the thickness of the primary xylem exhibited a relatively slow but continuous increase, consistent with the accumulation patterns of lignin and other substances during storage.” (Line218-225)
Additionally, we clarified the total number of samples in Section 2.3:
“To determine lignin content, a total of 40 samples were used, with four replicates for each of the ten time intervals.” (Line104-105) Since the primary focus of the study was on lignin content measurement and transcriptome analysis, we did not include additional replicates for anatomical sections at different time point. Therefore, the anatomical data serve as a trend-based references rather than detailed quantitative analyses.
2) since many genes are involved sensu lato in lignification however with very large (other?) functions as related in this paper (stress response, photosynthesis...) more or less far from your lignification focus, 2/ since the comparisons with pathways from other plants is quite criticized nowadays as these other plants correspond to other genomes and families, the relationship between genes and lignification is not clear and your conclusions should be much more cautious. Actually I do not see clearly the relationship between the phenotypes observed and the putative function(s) of the genes; there are (so) many correlations in plants but correlation and function are two different aspects not automatically connected; a correlation between gene and phenotype character (= lignification in your case) may exist, however the “real” function of a gene(s) is a very complex concept where “chain(s) of causation” has to demonstrate that for instance in your case lignification is caused by a given gene(s), which needs detailed physiology and anatomy and molecular (and etc…) studies, usually a very hard work!
Response:
We thank the reviewer for their insightful comments, particularly regarding the complexity of gene functions in lignification and the challenges of drawing conclusions from correlations with genes from other plant species. We agree that using pathways from other species to infer those of Moso bamboo may not be appropriate, as the genetic and functional contexts of different plant families can vary significantly. Additionally, we acknowledge that correlation between gene expression and phenotypic traits like lignification does not necessarily imply causation, and that establishing the true function of genes requires more detailed, direct experimental evidence. In response, we have made substantial revisions to the conclusion section. First, we removed imprecise statements, such as:
“It is speculating speculated that postharvest lignification in bamboo shoots is a self-protective mechanism under adverse environmental conditions like drought and cold, regulated by TFs, which promote the synthesis of JA and ABA. This, in turn, affects CAD enzyme activity, leading to increased lignin accumulation.” This statement has been revised to better reflect the tentative nature of our findings.
Second, we have revised the discussion section to enhance objectivity and logical coherence. The updated text now reads:
“The molecular mechanisms underlying lignification in Moso bamboo are highly intricate. Regulatory networks of senescence-associated genes, including transcription factors such as MYB and ERF, have been identified in bamboo shoots and shown to regulate pathways involved in lignin biosynthesis, highlighting their role in promoting degradation during postharvest storage [66]. The involvement of a hierarchical ubiquitination-mediated regulatory module further underscores the complexity of lignin biosynthesis regulation [67]. Key transcription factors, including MYB, play an active role in regulating lignin biosynthesis by modulating genes such as CAD and CCoAOMT. These findings offer a molecular framework for understanding the changes in lignin biosynthesis-related genes observed during postharvest storage [68]. Additionally, MYB can activate the expression of ABCG (ABC transporter G subfamily), which facilitates the transport of lignin monomers, thereby mediating lignin biosynthesis in Moso bamboo [69, 70].” (Line427-439)
We have also refined the discussion on methods to inhibit lignification, as shown in the revised statement:
“Shading to inhibit photosynthesis has been proposed as an effective strategy to delay lignification. This approach helps reduce the synthesis of phenolic acids, thereby decreasing the bitterness of bamboo shoots [55]. Moso bamboo subjected to heat treatment at 45°C, followed by storage at low temperatures, has been shown to effectively reduce weight loss, slow the decline in total sugar and acid content, mitigate browning, and delay the aging process of bamboo shoots [71]. Gamma-ray irradiation has also been found to suppress the activity of key enzymes (PAL and POD) involved in the lignin biosynthesis pathway, thereby regulating lignin and cellulose synthesis to slow lignification [72]. Melatonin treatment inhibits the activities of phenylalanine ammonia-lyase (PAL) and peroxidase (POD), effectively reducing lignification in fresh bamboo shoots [73]. Diphenyliodonium iodide inhibits NADPH oxidase activity, thereby slowing the postharvest hardening of bamboo shoots [74]. Additionally, treatments with oxalic acid [75], salicylic acid [76], and nitric oxide [77] have been shown to effectively delay lignification by regulating the activity of key enzymes involved in lignin biosynthesis.” (Line440-454)
3) Linked with point 1 above, 1/ for figure 2, since it is one of the bases of your article the photos are far too small, the sections are not enough contrasted and the differences between the tissues are almost not detectable; moreover put the value of the scale bar directly beside the scale, in order to be read more rapidly. 2/ for figure 3, the photos are also far too small, the contrasts are not enough enhanced and almost not visible in the sections: xylem should be much more green-blue, phloem light pink; moreover indicate that A-J are light microscopy (?); finally we do not see clearly which tissue corresponds to the (welcome) arrows. 3/ point 3.1. is not clear at all: the use of VA and PI is not understandable in terms of anatomy (refer to the terminology of types of cells and tissues as named in classical botanical anatomy; "the fresh bamboo shoots..." sentence has no verb ? "becoming largely inedible and losing their nutritional value" is an interpretation which has no anatomical support.
Response:
Regarding Figure 2:
Thank you for your comments. We have enhanced the contrast of Figure 2 to make the differences between tissues more distinguishable. Additionally, we moved the scale bar value next to the scale for quicker reading and better comprehension. Since Figure 2 primarily serves as a morphological reference to support the trends observed in subsequent experiments, we have removed it from the main manuscript and relocated it to Supplementary Figure S1.
Regarding Figure 3:
We have also adjusted the contrast of panels A–J in Figure 3 to improve the visibility of the details in the tissue sections. Furthermore, we have clearly indicated in the figure legend that panels A–J are light microscopy images. We have also refined the arrow annotations to ensure that they accurately point to the primary xylem, improving clarity. The updated text now reads as follows:
Figure 2. Changes in the anatomical structure of bamboo shoot vascular bundles and protoxylem during room temperature storage. (A)-(J) Changes in vascular bundles observed under light microscopy. (A) 0h, (B) 2h, (C) 4h, (D) 6h, (E) 8h, (F) 10h, (G) 12h, (H) 24h, (I) 48h, (J) 72h. (K)-(T) Changes in protoxylem observed under spontaneous fluorescence at the corresponding time points. (K) 0h, (L) 2h, (M) 4h, (N) 6h, (O) 8h, (P) 10h, (Q) 12h, (R) 24h, (S) 48h, (T) 72h. (PX) Protoxylem, (PP) Protophloem, (PC) Parenchyma, (SG) Starch grain. The arrow indicates the primary xylem. (Line208-213)
Regarding Section 3.1:
We appreciate your suggestions. To clarify, we have redefined VA and PI according to classical botanical anatomical terminology, specifically addressing cell types and tissue nomenclature. The revised sentence now reads: “with indistinct boundaries between the cortex (Co), vascular area (VA), and pith tissue (PT)”. (Line182-183)
Additionally, we have removed the phrases “the fresh bamboo shoots…” and “becoming largely inedible and losing their nutritional value” to make the conclusions and logic more precise and rigorous.
4) The PCA of figure 5 is far too weakly developed. As the PCA is just relating about the samples and not on the characters used for the matrix describing each sample, its interest is too weakly developed: It is usually of great interest to check about the importance of characters and etc…; if PCA is realized with supplemental data S3 (?), please detail each character contribution (line 2 of this S3) and their interest in your study. The percentage of contribution of each axis (1 and 2 I suppose?) I also very useful to state about the hierarchy between the characters and between the samples. Please check all these in mathematical tutorials easily found in internet.
Response: Thank you for your valuable suggestions. We have conducted a more detailed analysis of the PCA results, including a breakdown of the contribution of each feature to PC1 and PC2, and clarified their relevance in the context of our study. Additionally, we have specified the variance contribution ratios for both PC1 and PC2, which improves the hierarchical understanding of the relationships between samples. We have moved Figure 5 to Supplementary Figure S3, where the following changes have been incorporated:
(1) The percentage contribution of each principal component has been added.
(2) We have analyzed the relative importance of each PCA feature and discussed their significance.
(3) We’ve clarified the relationships and hierarchy among the samples, emphasizing the role of time points in shaping the gene expression profiles.
Specifically, we revised the manuscript as follows: “The PCA was performed based on the expression matrix of gene raw count values after TPM normalization. PC1 explained the majority of the variance, contributing 60% of the total variance. Notably, the clustering pattern revealed a distinct separation between samples from 0h and 72h, indicating that storage time has a profound impact on the gene expression profiles. Intermediate time points (such as 24h and 48h) displayed a gradual transition, reinforcing the progressive nature of the postharvest changes.” (Line247-253)
5) References already taken in account by the authors are of interest, however checking briefly in web of science WOS and scilit (from mdpi) with some key-words of this manuscript, (many…) other references appear for this largely studied topics and they should be updated and used (if relevant…) in order to sustain much more and provide a larger view of these researches.
Response: Thank you very much for your recommendation. conducted a thorough search using Web of Science (WOS) and Scilit (from MDPI) with relevant keywords related to the manuscript. As a result, we have supplemented and expanded the discussion section by incorporating additional references, which enhance the comprehensiveness and depth of our conclusions. These updated references provide a broader view of the research landscape and support our findings with more recent and relevant studies. Thank you for encouraging us to expand the reference list, and we believe these additional citations improve the quality of our manuscript.
Ref. 15. Yang, K.; Shan, X.; Shi, J.; Zhu, C.; Gao, Z. Identification and Expression Analysis of 4CL Gene Family in Phyllostachys edulis. Journal of Nuclear Agricultural Sciences 2021, 35, 72-82.
Ref. 16. Li, G.; Zhu, C.; Yang, K.; Wang, X.; Gao, Z. Identification and Expression Pattern Analysis of C4H Genes in Phyllostachys edulis. Journal of Tropical and Subtropical Botany 2022, 30, 151-160.
Ref. 66. Zhang, W.; Shi, M.; Yang, K.; Zhang, J.; Gao, Z.; El-Kassaby, Y.A.; Li, Q.; Cao, T.; Deng, S.; Qing, H.; et al. Regulatory networks of senescence-associated gene-transcription factors promote degradation in Moso bamboo shoots. Plant Cell Environ 2024, 47, 3654-3667, doi:10.1111/pce.14950.
Ref. 67. Yang, K.; Li, Z.; Zhu, C.; Liu, Y.; Li, H.; Di, X.; Song, X.; Ren, H.; Gao, Z. A hierarchical ubiquitination-mediated regulatory module controls bamboo lignin biosynthesis. Plant Physiology 2024, doi:10.1093/plphys/kiae480.
Ref. 68. Wang, B.; Chen, S.; Zhu, Z.; Li, Y.; Li, C.; Zhang, Y.; Huang, Y.; Xu, G.; Zhao, X.; Cao, Y.; et al. PeMYB26, an R2R3-MYB transcription factor, positively regulates lignin deposition in Moso bamboo. Plant Growth Regul 2024, doi:10.1007/s10725-024-01236-9.
Ref. 69. Li, Z.; Yang, K.; Zhu, C.; Liu, Y.; Guo, D.; Xiao, X.; Gao, Z. Identification and Expression Pattern Analysis of ABCG Genes in Moso Bamboo. Journal of Nuclear Agricultural Sciences 2023, 37, 917-926.
Ref. 70. Li, H.; Li, Z.; Yang, K.; Lin, Z.; Zhu, C.; Liu, Y.; Gao, Z. Comparison analysis of ABCG subfamily in bamboo and the potential function of PeABCG15 in monolignol transport. Plant Physiology and Biochemistry 2024, 217, doi:10.1016/j.plaphy. 2024.109278.
Minor points are:
1) As I am involved in plant taxonomy I am very sensible to correct plant taxa names which make their homogeneity and precision at the international level. In this respect the names of the author(s) (not in italics) have to be inserted (at least) the first time they appear in the text (from the beginning of the introduction), the whole text (especially for 2.7, see also for figure 8; see for all other parts) has to be revised carefully as there are mistakes. Use international Plant Names Index (IPNI) https://www.ipni.org/ or equivalent.
Response:
We appreciate the reviewer’s careful attention to plant taxonomy. In line with the recommendations, we have thoroughly revised the manuscript to ensure the accuracy and consistency of plant names according to the international standards. Specifically, we have used the International Plant Names Index (IPNI) to verify and update the taxonomic names throughout the manuscript. For instance, we have now correctly referenced Moso bamboo as Phyllostachys edulis (Carrière) J. Houz. in the introduction (Line 11) and have ensured that the full author citations are included the first time a plant species is mentioned.
Similarly, in Section 2.7 and other sections, we have corrected plant names and citations to reflect proper taxonomic standards, as follows: Bonia amplexicaulis (Thwaites) Stapleton, Guadua angustifolia Kunth, Olyra latifolia L., Raddia guianensis (Brongn.) Hitchc. (Line152-160)
2) Since palatability is not studied, I wonder if this word is relevant in the abstract, it would be better in the introduction or in the conclusion for further studies?
Response: We appreciate the reviewer’s observation regarding the use of the term "palatability." Since palatability was not directly studied in our research, we agree that it may be more appropriate to discuss it in the context of future research directions rather than the abstract. As a result, we have moved the mention of palatability to the discussion section, where we expand on its relevance for further studies. The revised text in the discussion is as follows:
“Photosynthesis during bamboo shoot storage can lead to greening and bitterness, while the phenylalanine biosynthesis pathway likely playing a key role in the formation of bitter chemical compounds. Related studies suggest that shading treatment postharvest can reduce the accumulation of phenolic acids associated with the tyrosine metabolism pathway, thereby reducing bitterness.” (Line409-413)
3) In 2.1, indicate in botanical terms the meaning of "the third section... excluding...", do you mean the third crown of leaf bases ? Indicate in full letters the meaning of FAA.
Response: Thank you for your valuable feedback. In response to your comment, we have clarified the meaning of "the third section" by providing a more detailed description of the sampling method. Additionally, we have spelled out the meaning of FAA for clarity. The revised text is as follows:
“To ensure uniformity among the experimental samples, starting from the node without bamboo roots at the base of the bamboo shoot, the third section from the bottom was horizontally cut to obtain a complete segment.” (Line81-83)
“one quarter was preserved in Formalin-Aceto-Alcohol (FAA) fixative solution for cytological observations.” (Line84-85)
4) For figure 1, change for "treatment", indicate for (a) the middle part and outer part of the section, (b) and (c) are not understandable in their precise location in the section.
Response: Thank you for your comment. We have made the requested changes in the figure legend and clarified the locations of the sections. Specifically, we have updated the term "treatment" to "treatment," as suggested, and added more detail to the figure description to better clarify the locations of the sections in panels (b) and (c). The revised text is as follows:
“After retaining the vascular tissues, the segment was divided into three parts.” (Line83-84)
5) In 2.2, indicate the meaning of "transparency", also the chemicals for staining. The sentence "Anatomical changes..." should be placed before "microphotographs were ..." ?
Response: Thank you for your suggestion. We have clarified the term “transparency” and provided more details regarding the staining procedure. The term "transparency" has been revised to "transparency treatment," and we have specified the chemical reagent used for staining as Safranin-O Fast Green staining solution. The revised text is as follows:
“Samples preserved in FAA were subjected to a series of steps including dehydration, transparency treatment, embedding, sectioning, staining with Safranin-O Fast Green solution, and mounting to create permanent sections [24,25].”(Line95-98)
Additionally, we have rearranged the sentence order for better flow, moving "Anatomical changes..." to appear before "Microphotographs were captured..."
6) For figure 4, indicate if it is the standard deviation of confidence interval which is indicated for each mean.
Response: Thank you for your suggestion. We have clarified that the error bars represent the standard deviation. The updated description in the figure legend now reads: “The error bars in the figure represent the standard deviation.” (Line227-228)
7) For figure 5 B, indicate what means the horizontal and vertical axes; for the PCA A, indicate the meaning of each of the three sets of data.
Response: Thank you for your valuable correction. We have added clarifications for the horizontal and vertical axes in Figure 5B (now as figure S3). We have also clarified the meaning of each of the three data sets shown in the PCA plot. The revised explanation in the article now reads: “The results indicated that the 24 samples were grouped into three main clusters based on their relative expression levels (TPM): samples from 0-6h formed one group, 12-24h formed another, and 48-72h formed a third group.” (Line253-255)
8) Restrict results to only raw data and delete all sentences belonging actually to the discussion ("suggesting" in 3.3," indicate" in 3.4., "may be attributed" in 3.5; see for all other parts.
Response: We appreciate your suggestion and have carefully revised the manuscript and removed sentences that belong to the discussion section, as per your recommendation.
9) For figure 6, put all your texts in bold letters, they are almost invisible as too small; precise the meanings of the scales as the very short title used in not understandable; indicate also in full letters DEG and GO, a figure has to be understood by itself, not referring to the text.
Response: Thank you for your valuable feedback. We have made the following revisions to Figure 6: (1) All text in the figure has been changed to bold font to improve visibility and readability. (2) The meaning of the scale is now clearly stated in the figure legend. (3) We have expanded the abbreviations for DEG (Differentially Expressed Genes) and GO (Gene Ontology) to ensure the figure can be understood independently, without referring to the main text. The revised figure legend now reads: “Description of differentially expressed genes (DEGs) analysis. GO analysis of specifically expressed genes in shoots. (A) 0h. (B) 4h. (C) 12h. (D) 48h. (BP) Biological Process. (CC) Cellular Component. (MF) Molecular Function. The color scale indicates the P-value and the dots represent the number of genes enriched in the pathway.” (Line273-276)
10) For figure 7, for the same reasons indicate in full letters in B the meaning of WGCNA and DEG; indicate also the meaning of the colors; moreover B is far too small and almost invisible.
Response: Thank you for your constructive comments. We have made the following revisions to Figure 7: (1) We have expanded the abbreviations for WGCNA (Weighted Gene Co-expression Network Analysis) and DEG (Differentially Expressed Genes) to ensure clarity. (2) The meaning of the color scales has been clearly indicated in the figure legend, and the significance of the colors has been explained. (3) We have enlarged the elements in Panel B to improve their visibility and readability, as you pointed out. The updated figure legend now reads: “Weighted Gene Co-expression Network Analysis (WGCNA) of identified differentially expressed genes (DEGs). (A) Module-trait relationships. (a) Storage times. (b) Lignin content. (c) Autofluorescence content of vascular bundles. (d) Autofluorescence content of the PX. The scale indicates the correlation between the module and trait. (B) Gene expression profile of the brown module. The color scale indicates the relative expression of genes. (C) The protein interaction regulatory network in the brown module, with transcription factors highlighted in purple.” (Line299-304)
11) For figure 8, use the same format of letters for each taxon; moreover if the values correspond to former articles indicate them.
Response: Thank you for your comment. We have standardized the font format for each taxon in Figure 8 to ensure consistency.
12) For figure 9, indicate in full letters the meaning of DEG.
Response: Thank you for your suggestion. We have spelled out the meaning of DEG as "differentially expressed genes" and clarified the color scale in the figure legend. The updated description is as follows: “Heat map depicting the expression profiles of differentially expressed genes (DEGs) involved in lignin biosynthesis in Moso bamboo. The color scale indicates the relative expression of genes. The heatmap shows the expression trend of some enzyme genes at different storage times.” (Line350-352)
Thank you very much for your consideration.
Sincerely,
Chunce Guo
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsComments to authors
The manuscript ID: forests-3376611 entitled ‘’Lignification and Gene Expression Pattern in Postharvest Moso Bamboo Shoots’’ by Huang et al. analyzed morphological changes, cellular alterations, lignin content, and gene expression at various postharvest time points to investigate the changes in lignin accumulation. Additionally, the study aimed to determine the maximum storage duration allowed at room temperature for preservation, with the ultimate goal of proposing effective preservation methods. I consider the idea and intention of the article to be good and reported important results. Below you can find to be considered the most relevant:
Major points:
- Page 5, line 182: Please revise the sentence ‘’The fresh bamboo shoots, no significant morphological changes were observed within the first 12 hours of placement.’’ There is a contradiction with the previous sentence.
- In relation with the dynamic expression of internal genes in Moso bamboo shoots during postharvest storage at room temperature, the authors conducted transcriptome sequencing and they excluded relatively unchanged time points at 8h and 10h. My question is why you didn’t exclude also the time point of 6h as the plateau period is included between 4h and 12h?
- The same comment for the Venn diagram of DEGs? The authors exclude the point time 72h.
- The JA pathway is known to be related to biotic stress. How to explain it role in the lignification of Moso bamboo shoots during postharvest storage?
Minor points
- Legend of Figure 1: Please change the word ‘’ treantment’’ to ‘’treatment’’.
- Legend of Figure 1: Revise as follow: (c) Section used for transcriptomic analysis.
- Please define the abbreviation of ‘’FAA’’.
- Page 4, line 133, delete the word : ‘’the’’ before the sentence ‘’GO database terms’’.
- The name of genes must be in italic. Please check the entire manuscript.
Author Response
Dear Reviewer,
We greatly appreciate your comments and suggestions. We are especially grateful for your encouragement to submit a revised manuscript and for your highlight areas of revisions. We have carefully reviewed all of your comments and made the necessary revisions in the updated version. The comments are reproduced, and our responses follow directly after each comment.
The manuscript ID: forests-3376611 entitled ‘’Lignification and Gene Expression Pattern in Postharvest Moso Bamboo Shoots’’ by Huang et al. analyzed morphological changes, cellular alterations, lignin content, and gene expression at various postharvest time points to investigate the changes in lignin accumulation. Additionally, the study aimed to determine the maximum storage duration allowed at room temperature for preservation, with the ultimate goal of proposing effective preservation methods. I consider the idea and intention of the article to be good and reported important results. Below you can find to be considered the most relevant:
Response: We sincerely thank the reviewer for the positive evaluation of our work and greatly appreciate the valuable suggestions provided to further improve it.
Major points:
- Page 5, line 182: Please revise the sentence “The fresh bamboo shoots, no significant morphological changes were observed within the first 12 hours of placement.’’ There is a contradiction with the previous sentence.
Response: Thank you very much for your valuable feedback. Upon review, we agree that the sentence contradicts the previous sentence. We have revised and removed certain sentences to ensure consistency and clarity. For example, we have updated the text to: "The transverse sections of Moso bamboo shoots stored at different time intervals displayed varying degrees of morphological changes" (Line180-181) and have also delete the sentence "the changing trends of these three components are the same".
- In relation with the dynamic expression of internal genes in Moso bamboo shoots during postharvest storage at room temperature, the authors conducted transcriptome sequencing and they excluded relatively unchanged time points at 8h and 10h. My question is why you didn’t exclude also the time point of 6h as the plateau period is included between 4h and 12h?
Response: Thank you for your question. The 6 -hour time point was not excluded because it marks the transition from the early growth period to the plateau phase, with the plateau period spanning from 6 hours to 12 hours. We selectively excluded the two middle time points within the plateau phase (8 hours and 10 hours), as they showed minimal variation. We decided to retain the 6-hour time point to capture the early onset of the plateau period, ensuring a more comprehensive representation of gene expression dynamics.
- The same comment for the Venn diagram of DEGs? The authors exclude the point time 72h.
Response: Thank you for your comment. The 72-hour time point was excluded from the differential expression gene (DEGs) Venn diagram because preliminary analysis revealed that the gene expression patterns at 72 hours were highly similar to those at 48 hours. Likewise, we observed almost identical gene expression trends at the 2-hour and 4-hour time points, as shown in the sample correlation heatmap (Figure S3 B). To avoid the potential confounding effect of sample similarity and to ensure a more accurate comparison of specifically expressed genes at each time point, we chose not to include the 2-hour and 72-hour time points in the Venn diagram.
- The JA pathway is known to be related to biotic stress. How to explain it role in the lignification of Moso bamboo shoots during postharvest storage?
Response: Thank you for your insightful question. The Jasmonic acid (JA) pathway is widely known for its role in plant defense responses, developmental processes, and adaptation to environmental stresses. In the context of our study, which focuses on the lignification of bamboo shoots during post-harvest storage at room temperature, bamboo shoots are subjected to various stresses such as drought, temperature fluctuations, and potential pest damage. These stresses may lead to the passive activation of the JA pathway.
We identified a significant enrichment of JA pathway-related genes among those specifically expressed at the 12-hour time point, which coincides with a rapid increase in lignification. Additionally, through weighted gene co-expression network analysis (WGCNA), we found that LOX proteins, which are involved in JA biosynthesis, were present in the brown module and showed the highest correlation with lignin content. These findings suggest a potential role for JA in the postharvest lignification process of bamboo shoots. However, we acknowledge that it is challenging to directly infer the metabolic pathways of bamboo shoots based on those of other species, and correlating these findings with functional analysis may be somewhat speculative.
To address this, we have revised the conclusion section. We removed imprecise statements, such as: “It is speculated that postharvest lignification in bamboo shoots is a self-protective mechanism under adverse environmental conditions like drought and cold, regulated by TFs, which promote the synthesis of JA and ABA. This, in turn, affects CAD enzyme activity, leading to increased lignin accumulation.”
Further, we expanded the discussion by referencing additional literature to enhance the objectivity and logical coherence of the analysis. we also included references to practical approaches, such as: “The molecular mechanisms underlying lignification in Moso bamboo are highly intricate. Regulatory networks of senescence-associated genes, including transcription factors such as MYB and ERF, have been identified in bamboo shoots and shown to regulate pathways involved in lignin biosynthesis, highlighting their role in promoting degradation during postharvest storage. The involvement of a hierarchical ubiquitination-mediated regulatory module further underscores the complexity of lignin biosynthesis regulation. Key transcription factors, including MYB, play an active role in regulating lignin biosynthesis by modulating genes such as CAD and CCoAOMT. These findings offer a molecular framework for understanding the changes in lignin biosynthesis-related genes observed during postharvest storage. Additionally, MYB can activate the expression of ABCG (ABC transporter G subfamily), which facilitates the transport of lignin monomers, thereby mediating lignin biosynthesis in Moso bamboo.” (Line427-439) and “Shading to inhibit photosynthesis has been proposed as an effective strategy to delay lignification. This approach helps reduce the synthesis of phenolic acids, thereby decreasing the bitterness of bamboo shoots. Moso bamboo subjected to heat treatment at 45°C, followed by storage at low temperatures, has been shown to effectively reduce weight loss, slow the decline in total sugar and acid content, mitigate browning, and delay the aging process of bamboo shoots. Gamma-ray irradiation has also been found to suppress the activity of key enzymes (PAL and POD) involved in the lignin biosynthesis pathway, thereby regulating lignin and cellulose synthesis to slow lignification. Melatonin treatment inhibits the activities of phenylalanine ammonia-lyase (PAL) and peroxidase (POD), effectively reducing lignification in fresh bamboo shoots. Diphenyliodonium iodide inhibits NADPH oxidase activity, thereby slowing the postharvest hardening of bamboo shoots. Additionally, treatments with oxalic acid, salicylic acid, and nitric oxide have been shown to effectively delay lignification by regulating the activity of key enzymes involved in lignin biosynthesis.” (Line440-453).
Minor points:
- Legend of Figure 1: Please change the word “treantment’’ to “treatment’’.
Response: Thank you for your correction. We have updated the word "treantment" to "treatment" in the legend of Figure 1.
- Legend of Figure 1: Revise as follow: (c) Section used for transcriptomic analysis.
Response: Thank you for your suggestion. We have revised the legend as requested to: "(c) Section used for transcriptomic analysis." (Line93)
- Please define the abbreviation of “FAA’’.
Response: Thank you for pointing that out. We have now spelled out the abbreviation “FAA” in full. The revised sentence is: “One quarter was preserved in Formalin-Aceto-Alcohol (FAA) fixative solution for cytological observations.” (Line84-85)
- Page 4, line 133, delete the word: “the’’ before the sentence “GO database terms’’.
Response: Thank you for your suggestion. We have removed the word “the” before “GO database terms” as requested.
- The name of genes must be in italic. Please check the entire manuscript.
Response: Thank you for your helpful suggestion. We have carefully reviewed the manuscript and italicized all gene names as required.
Thank you very much for your consideration.
Sincerely,
Chunce Guo
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsComments: Very good study. Whther biological replicates are used for this transcriptome analysis stydy?
Line 24-26, Very long sentence with many times use of ‘and’
At line 27, Whether Bamboo shoots are highly prized delicacies in China and other
Asian countries? Write clearly about which type of shoots?
Discus more about enrichment of genes at different hours of postharvest. Why those genes are enriching?
Discuss your results comparing with earlier findings.
Improve conclusion
Reduce similarity with published words and sentences.
Enlish grammer and uses need to be rechecked.
Comments on the Quality of English Languagescope for improvement
Author Response
Dear Reviewer,
Thank you for the comments concerning our manuscript entitled "Lignification and Gene Expression Pattern in Postharvest Moso Bamboo Shoots" (ID: forests-3376611) Those comments are very valuable and helpful for improving our paper and have important guiding significance to our research. We have studied the comments carefully and modified the manuscript point-by-point. The responses to the comments are as follows:
Very good study. Whther biological replicates are used for this transcriptome analysis stydy?
Response: Thank you for pointing this out. We have supplemented this information in Section 2.4 as follows: “Samples were sequenced at eight time points: 0h, 2h, 4h, 6h, 12h, 24h, 48h, and 72h, with three replicates for each time point.” (Line116-117)
Line 24-26, Very long sentence with many times use of ‘and’.
Response: Thank you for your suggestion. We have simplified and divided the sentence as follows: “Moso bamboo (Phyllostachys edulis (Carrière) J. Houz.) covers an area of approximately 30,000 km². It is one of the most important bamboo species in China. This species serves as a primary source for both bamboo timber and bamboo shoot production and plays a crucial role in the ecological environment.” (Line24-27)
At line 27, At line 27, Whether Bamboo shoots are highly prized delicacies in China and other Asian countries? Write clearly about which type of shoots?
Response: Thank you for bringing this to our attention. As the sentence was not clearly expressed, we have decided to delete it.
Discus more about enrichment of genes at different hours of postharvest. Why those genes are enriching?
Response: Thank you for your question. The specific enrichment results are presented in Figure 4. As these results cannot be interpreted subjectively, we have selected certain interesting pathways from the GO enrichment analysis and elaborated on them in the discussion section.
For example:
“Environmental stress potentially contributes to rapid lignification. Transcriptome sequencing revealed that genes specifically expressed at 4 hours postharvest were enriched in pathways related to responses to water deficit and temperature stress. Drought conditions likely influence enzyme activity in the lignin biosynthesis pathway, thereby accelerating lignification. Temperature also plays a role in lignification, as demonstrated in species like loquat, and kiwi, where low temperature regulate lignification.”(Line397-403)
Additionally:
“Bamboo shoots remain metabolically active postharvest, undergoing vigorous metabolic activities. At 12 hours postharvest, pathways related to abscisic acid (ABA) response and photosynthesis were significantly enriched. ABA is a key hormone mediating physiological responses to various environmental stress, including drought, salinity, cold, and pathogen.” (Line404-408)
Discuss your results comparing with earlier findings.
Improve conclusion.
Response: Thank you for your suggestion. We have significantly revised the conclusion section to improve precision, coherence, and depth. Firstly, we removed imprecise statements, such as “It is speculating that postharvest lignification in bamboo shoots is a self-protective mechanism under adverse environmental conditions like drought and cold, regulated by TFs, which promote the synthesis of JA and ABA. This, in turn, affects CAD enzyme activity, leading to increased lignin accumulation.”
Secondly, we enriched the discussion by referencing additional literature, enhancing the objectivity and logical flow. The revised conclusion is as follows: “The molecular mechanisms underlying lignification in Moso bamboo are highly complex. Regulatory networks of senescence-associated genes, including transcription factors such as MYB and ERF, play crucial roles in regulating pathways involved in lignin biosynthesis, thereby promoting degradation during postharvest storage. The hierarchical ubiquitination-mediated regulatory module further underscores the intricacy of lignin biosynthesis regulation. Key transcription factors such as MYB not only regulate lignin biosynthesis -related genes, including CAD and CCoAOMT , but also activate the expression of the ABC transporter G subfamily (ABCG), facilitating lignin monomer transport and mediating lignin biosynthesis. These findings offer a molecular framework for understanding the dynamic changes in lignin biosynthesis-related genes during postharvest storage.” (Line427-439)
Additionally, we integrated practical approaches to delay lignification:
“ Shading to inhibit photosynthesis has been proposed as an effective strategy to delay lignification. This approach helps reduce the synthesis of phenolic acids, thereby decreasing the bitterness of bamboo shoots [55]. Moso bamboo subjected to heat treatment at 45°C, followed by storage at low temperatures, has been shown to effectively reduce weight loss, slow the decline in total sugar and acid content, mitigate browning, and delay the aging process of bamboo shoots [71]. Gamma-ray irradiation has also been found to suppress the activity of key enzymes (PAL and POD) involved in the lignin biosynthesis pathway, thereby regulating lignin and cellulose synthesis to slow lignification [72]. Melatonin treatment inhibits the activities of phenylalanine ammonia-lyase (PAL) and peroxidase (POD), effectively reducing lignification in fresh bamboo shoots [73]. Diphenyliodonium iodide inhibits NADPH oxidase activity, thereby slowing the postharvest hardening of bamboo shoots [74]. Additionally, treatments with oxalic acid [75], salicylic acid [76], and nitric oxide [77] have been shown to effectively delay lignification by regulating the activity of key enzymes involved in lignin biosynthesis.” (Line440-453)
Reduce similarity with published words and sentences.
Response: Thank you for the feedback. To address this, we have revised the manuscript to minimize the similarity with previously published content. This involved rephrasing key sections, adopting alternative terminology, and restructuring sentences to ensure originality while preserving the clarity and accuracy of the scientific information presented.
Enlish grammer and uses need to be rechecked.
Response: Thank you for highlighting this issue. We have thoroughly reviewed the manuscript for grammatical errors and have made the necessary corrections to improve language accuracy and clarity. We are confident that the revised version adheres to standard English grammar and usage.
Thank you once again for your thoughtful review and consideration.
Sincerely,
Chunce Guo
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsDear authors,
I read with a great interest this revision, however still some points have to be clarified and a major revision is (still) requested, mainly for the first very important item.
Sincerely yours,
For my major point 1, measurements (supplements 2 and 3) are welcome, however we need t student tests (or equivalent for 40 data, as I suggested in my first review [“then you can proceed to statistical analyses (for instance t student tests…”] to state if the differences are or not significantly different for each stage evaluated (please provide the diagrams for each test). Both significant differences or un-significant (= tendencies) differences are of biological interest. Your sentence “Specifically, the area and diameter… » has no precise meaning and just looking at your matrix I suspect (?) that some values are (?) significantly different while others are not (which has another meaning in your biological interpretation(s)), this will give a real objective and necessary value to your article. Moreover, for statistics, generally speaking confidence interval is much more relevant, argument if you still wish to keep standard deviation. Finally for supplements 3, precise the units of measurements of each column and what is taken in account in the proportion column.
For my major point 2, your sentence “particularly regarding the complexity of gene functions in lignification and the challenges of drawing conclusions from correlations with genes from other plant species” should appear in your conclusion as it is exactly what happens in many plants, your updated text is too strong.
For my major point 3, as you removed one figure divide figure 2 in two sub-figures as it is still unclear. In 2.1, as you used 2/4 of the sections, what about the half part remaining?
Author Response
Dear Reviewer,
Thank you once again for your insightful comments and valuable suggestions. We deeply appreciate the time and effort you have invested in reviewing our manuscript. Your thoughtful feedback has been instrumental in refining and strengthening our work.
In response to your most recent review, we have made significant revisions to the manuscript to enhance both the clarity and depth. Below, we provide a point-by-point response addressing your specific comments. We sincerely hope these revisions meet your expectations and satisfactorily address the concerns you have raised.
For my major point 1, measurements (supplements 2 and 3) are welcome, however we need t student tests (or equivalent for 40 data, as I suggested in my first review [“then you can proceed to statistical analyses (for instance t student tests…”] to state if the differences are or not significantly different for each stage evaluated (please provide the diagrams for each test). Both significant differences or un-significant (= tendencies) differences are of biological interest. Your sentence “Specifically, the area and diameter… » has no precise meaning and just looking at your matrix I suspect (?) that some values are (?) significantly different while others are not (which has another meaning in your biological interpretation(s)), this will give a real objective and necessary value to your article. Moreover, for statistics, generally speaking confidence interval is much more relevant, argument if you still wish to keep standard deviation. Finally for supplements 3, precise the units of measurements of each column and what is taken in account in the proportion column.
Response: Thank you for your thoughtful feedback, which have significantly helped improve the quality of our manuscript. We have carefully addressed your comments as follows:
(1) Statistical Analyses: As you suggested, we performed t-tests on the measurement data in Supplementary Table 2 to determine whether the differences between stages were statistically significant. The updated results are now included in Supplementary Table S3 and and visually represented in Figure S2. Specifically, we expanded the discussion to include these findings: “To evaluate the significance of changes in the content of various substances between adjacent time points, we conducted t-tests for the three substances (Supplementary Table S3) and visualized the trends using bar charts (Figure S2). The results revealed that during the early storage period (0h to 4h), the differences between time points were not statistically significant (P > 0.05). However, in the later stages of storage (12h to 24h and 48h to 72h), the differences became significant (P < 0.05). By contrast, autofluorescence content, measured both in vascular bundles and primary xylem, exhibited significant differences across all time points, with extremely low P-values (< 0.00001), particularly during transitions from 12h to 24h and 48h to 72h.” (line 220-228)
(2) Morphological Changes of the Primary Xylem: To provide a more accurate interpretation, we conducted linear regression analysis on the data from Supplementary Table S4 and calculated the fitted curves for each change. These results are now presented in Supplementary Table S5 and Figure S4. We have revised the discussion of these findings as follows: “A linear regression model was employed to generate the fitted curves for the primary xylem data (Supplementary Table S5). The analysis revealed that the area, thickness, and diameter of the primary xylem exhibited significant changes during storage. The area initially decreased (0h to 6h) but then increased rapidly, with the growth rate gradually slowing but continuing to rise. The thickness and diameter showed a consistent increase during the early storage period, slight fluctuations in the mid-stage, and resumed growth in the later stages. Overall, the primary xylem parameters demonstrated significant changes during the early storage period, followed by a period relatively steady growth (Figure S4).” (line 231-239)
Additionally, we removed the previously inaccurate statement: “Specifically, the area and diameter of the primary xylem decreased rapidly from 0 to 4 hours, then gradually recovered after 6 hours, showing a significant increase by 12 hours. In contrast, the thickness of the primary xylem exhibited a relatively slow but continuous increase, consistent with the accumulation patterns of lignin and other substances during storage.”
(3) Standard Deviation vs. Confidence Interval: After careful consideration, we decided to retain the use of standard deviation in the manuscript. The standard deviation is a concise and easily interpretable statistic, suitable for our descriptive analysis and the primary focus of this study, which is to explore data variability. While confidence intervals are more relevant for inferential statistics, our study emphasizes the dispersion and variability of the data, making standard deviation a more appropriate choice.
Supplementary Table S3 Updates: We have clarified the units of measurement for each column and the content considered in the proportion column. The updated table now specifies: “Primary xylem area (nm2), Primary xylem diameter (nm), Primary xylem thickness (nm), The proportion of primary xylem in vascular bundles (%).”
For my major point 2, your sentence “particularly regarding the complexity of gene functions in lignification and the challenges of drawing conclusions from correlations with genes from other plant species” should appear in your conclusion as it is exactly what happens in many plants, your updated text is too strong.
Response: Thank you for your insightful suggestion. We have carefully considered your point and have integrated this content into the discussion section to emphasize the limitations of our study. The revised text reads as follows: “However, because the genetic and functional contexts of different plant families can vary significantly, using pathways from other species to infer those of Moso bamboo may not be entirely appropriate. This is particularly relevant given the complexity of gene functions in lignification and the inherent challenges in drawing definitive conclusions from correlations with genes identified in other plant species.” (line 441-446)
For my major point 3, as you removed one figure divide figure 2 in two sub-figures as it is still unclear. In 2.1, as you used 2/4 of the sections, what about the half part remaining?
Response: Thank you for your suggestion. Since there are ten time points, each represented by a small plot, the only feasible layout options are 2x5 or 5x2. For aesthetic and clarity purposes, we decided not to split them into two subplots. Instead, we have re-exported the images in higher resolution and adjusted their size in the manuscript. The cell structures are now clearly visible.
Regarding Section 2.1, we have added the following clarification: “One quarter of the sample was preserved in Formalin-Aceto-Alcohol (FAA) fixative solution for cytological observations, another quarter was placed in 15 mL centrifuge tubes and stored at -80°C for subsequent transcriptomic analysis, and the remaining half was used for lignin content measurement (Figure 1)” (line 84-88)
Thank you again for your support and constructive input throughout the review process.
Sincerely,
Chunce Guo
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsAll points raised were well reviewed by the authors. The manuscript is now improved and can be published in the journal ''Forests''.
Author Response
Dear Reviewer,
Thank you for your thorough and thoughtful feedback, as well as for accepting our manuscript for publication. We sincerely appreciate your constructive suggestions, which have greatly contributed to improving the clarity and rigor of our work. We have carefully addressed all the comments and implemented the necessary revisions to enhance the overall quality of the manuscript. We are confident that the revised version aligns with the publication standards. Once again, we are grateful for your time and effort in reviewing our work.
Sincerely,
Chunce Guo
Round 3
Reviewer 1 Report
Comments and Suggestions for AuthorsDear authors,
This version is now fine to me and I think it is a good work with quite rare anatomical data related with molecular biology. A last minor point concerns the letters of the A-J photos of figure 2 which should be more visible in black, white letters being almost invisible for some of them.
Very sincerely yours,
Author Response
Dear Reviewer,
Thank you once again for your insightful comments and valuable suggestions. We deeply appreciate the time and effort you have invested in reviewing our manuscript. Your thoughtful feedback has been instrumental in refining and strengthening our work.
In response to your most recent review, we have made the minor revisions you requested to further improve the manuscript. Below, we provide a concise, point-by-point response addressing your comments:
This version is now fine to me and I think it is a good work with quite rare anatomical data related with molecular biology. A last minor point concerns the letters of the A-J photos of figure 2 which should be more visible in black, white letters being almost invisible for some of them.
Response: Thank you very much for your valuable suggestion. We have addressed this issue by adjusting the photos in Figure 2 (A-J) to improve visibility. We hope this enhances the clarity of the figure for better readability.
Once again, thank you for your thoughtful consideration.
Sincerely,
Chunce Guo
Author Response File: Author Response.pdf
