Genome-Wide Identification and Expression Analysis of the Trehalose-6-phosphate Synthase and Trehalose-6-phosphate Phosphatase Gene Families in Rose (Rosa hybrida cv ‘Carola’) under Different Light Conditions

Response to Reviewer 1 Comments

1. Summary

Thank you very much for your comments and professional advice. Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. For clarity, we will use red text to show our revisions.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1:

This study used the old software MEGA X for Phylogenetic analysis. I think it's better to use the latest version of MEGA 11.

Response 1:

Thank you very much for your attention and suggestions on our research. After team discussion, we fully agree with your suggestion and will use MEGA 11 for phylogenetic analysis in the revised version. In actual use, we also found that the running speed of MEGA11 has been improved compared to MEGAX. The specific changes are shown below, and we have also redrawn the evolutionary tree. We hope to receive your approval.

4.4. Phylogenetic relationships, gene structure, conserved motif and cis-Element analysis

The TPS and TPP phylogenetic relationships of R. hybrida, A. thaliana, Populus trichocarpa, Malus domestica, Fragaria vesca, R. rogusa and R. wichurana were reconstructed by MEGA11 [55] with the muscle program to perform multiple sequence alignments and Maximum Likelihood Method (ML) analyses, with the bootstrap value set at 1000 repetitions.

55. Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Molecular biology and evolution 2021, 38, 3022-3027, doi:10.1093/molbev/msab120.

Figure 2. Construction of phylogenetic tree proteins for TPS and TPP.

(a) Phylogenetic tree of the TPS proteins from seven plant species. The TPS pro-teins of R. hybrida (RhTPS), P. trichocarpa (PtTPS), A. thaliana (AtTPS), Malus domestica (MdTPS), Fragaria vesca (FvTPS), Rosa rugosa (RrTPS) and Rosa wichruana (RwTPS).

(b) Phylogenetic tree of the TPP proteins from seven plant species. The TPP proteins of R. hybrida (RhTPP), P. trichocarpa (PtTPP), A. thaliana (AtTPP), M. domestica (MdTPP), F. vesca (FvTPP), R. rugosa (RrTPP) and R. wichruana (RwTPP).

Comments 2:

  This paper applies stress using different light conditions, but there is a similar paper that studies differences in gene expression due to heat stress. Xiao-Ru Wei et al., Horticulturae, 2022, 8, 429. https://doi.org/10.3390/horticulturae8050429

Response 2:

We are very grateful for the reviewer's inquiries. Wei et al. have made significant contributions to the identification and analysis of the rose TPS gene family, particularly in studying the impact of different heat stress conditions on TPS gene expression. However, we simultaneously identified and analyzed two gene families involved in trehalose synthesis, TPS and TPP, rather than focusing solely on one TPS gene family. Because plants synthesize trehalose through a conserved, two-step metabolic pathway. The first step involves the conversion of glucose from UDP-glucose to glucose 6-phosphate by trehalose-6-phosphate synthase (TPS), resulting in the production of treha-lose-6-phosphate (T6P). The T6P is then dephosphorylated into trehalose by trehalose 6-phosphate phosphatase (TPP). Furthermore, trehalose has been extensively reported to participate in plant responses to both high and low temperatures [19]. Especially, our research has uniqueness in addressing the differential gene expression under light conditions.

Specifically, there are several differences between our article and Wei et al.'s article:

Firstly, our study not only identified one gene family, but also simultaneously identified members of two gene families and studied their expression patterns. More and more studies are simultaneously focusing on the expression patterns of two gene families in response to stress in plants [27]. Our study is the first to simultaneously identify and analyze the genes of TPS and TPP family members in roses, which can better characterize the mode of action of the fucose metabolism pathway in roses and provide a more detailed basis for future research. This comprehensive study can provide more comprehensive gene family information, helping us better understand the functions and interactions of trehalose related gene families in organisms.

Secondly, the methods and logic we analyzed differ significantly from Wei's paper. One reason is that the scientific issues we focus on are inconsistent. We focus on the flowering phenotype of roses, and during the sampling process, we take stem tip meristems closely related to flowering. Wei et al. focused on the heat tolerance of rose cuttings, so the sampling time span was shorter than our study. And we provided the phenotypic changes of the processed roses in our paper, which were not reflected in Wei et al.'s paper. There are also differences in specific analysis methods. The genome data of roses was published in 2017, and with the development of bioinformatics technology, gene structure annotations have been manually corrected. In this article, we employed the GSAman tool (https://gitee.com/cjchen/igv-srna) to rectify the gene structure of TPS and TPP gene family members prior to conducting our analysis. It is believed that this is not only the first accurate identification and analysis of the rose TPP gene family, but also the enrichment and improvement of the rose TPS family. In addition, we analyzed the expression patterns of identified TPS and TPP gene families using both transcriptome data and qPCR expression level data. In terms of specific expression patterns, we simultaneously investigated tissue-specific expression and light-induced expression after environmental treatment, resulting in a more comprehensive and detailed dataset. In addition, when analyzing the promoter regions, we pay more attention to the specific types of cis acting elements, while Wei focuses on the functions. A recently published article confirms our accurate prediction of the interaction between the TPP family and myb transcription factors [38]. We will also update in the paper.

Finally, based on previous observations and measurements of flowering phenotypes and indicators in roses under different light environments, our study found that trehalose may also be involved in the flowering response of roses under low light conditions, which has never been reported in roses, especially since the combined involvement of light and sugar signals in the flowering process of roses has not yet been revealed. Unlike the TPS gene family reported separately by Wei et al., we focus more on the key roles of TPS, TPP, and Tre in the flowering of roses under light environments.

We will also present our method, experimental design, and results in detail in the paper, and compare and discuss them with existing related studies to highlight the uniqueness and contribution of our research. Thank you again for the guidance and feedback from the reviewer.

[19] Raza, A.; Bhardwaj, S.; Atikur Rahman, M.; García-Caparrós, P.; Habib, M.; Saeed, F.; Charagh, S.; Foyer, C.H.; Siddique, K.H.M.; Varshney, R.K. Trehalose: A sugar molecule involved in temperature stress management in plants. The Crop Journal 2023, doi:https://doi.org/10.1016/j.cj.2023.09.010.

[27] Gao, Y.H.; Yang, X.Y.; Yang, X.; Zhao, T.Y.; An, X.M.; Chen, Z. Characterization and expression pattern of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase gene families in Populus. International Journal of Biological Macromolecules 2021, 187, 9-23, doi:10.1016/j.ijbiomac.2021.07.096.

[38] Yu, H.; Teng, Z.; Liu, B.; Lv, J.; Chen, Y.; Qin, Z.; Peng, Y.; Meng, S.; He, Y.; Duan, M.; et al. Transcription factor OsMYB30 increases trehalose content to inhibit alpha-amylase and seed germination at low temperature. Plant physiology 2023, doi:10.1093/plphys/kiad650.

Comments 3:

Response to Reviewer 2 Comments

1. Summary

Dear Reviewer, thank you very much for taking the time to review our manuscript. We greatly appreciate your professional insights and valuable suggestions. Below, we have provided detailed responses to your comments and made corresponding revisions and corrections in the resubmitted files. We sincerely hope that these modifications meet your expectations and enhance the quality of our research. For clarity, we will use red text to show our revisions.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1:

The last paragraph of the introduction should contain the aim of the study. Instead, the writers gave the need for work followed by the conclusion. There is no aim, objective, or scope of the study.

Response 1:

Thank you very much for your question and helping us improve the quality of our manuscript. As suggested, we have rewritten the last paragraph of the introduction to emphasize our research objectives, aims, and scope, enabling readers to accurately understand our research intent. This change can be found in the revised version of the article (line84-99). The specific modifications are as follows:

Rosa hybrida is currently the largest cut flower in terms of global planting area and sales volume [18]. The TPS and TPP gene families have been reported to be of great significance in many kinds of plants response to stress and regulation of growth and development [19,20]. With the update of gene structure annotation technology(https://gitee.com/CJchen/IGV-sRNA), and so rose TPS genes need be reidentified and analyzed. Furthermore, there have been no studies to evaluate the evolution and expression profiles of the TPP gene family in Rosa hybrida. In this study, the member of TPS and TPP family were identified in rose, and their structures, phylogenetic relationships，cis-acting elements were thoroughly analyzed. The expression patterns of TPS and TPP in different tissues of roses were analyzed using transcriptome data. Additionally, the crucial role of RhTPS and RhTPP in flower formation in response to various photoperiods and light intensities were investigated by transcriptome data and qRT-PCR. Finally, we conducted a correlation analysis between flowering time, trehalose content, and RhTPSs/TPPs gene expression to identify key functional genes. The findings from this study serve as a valuable biological foundation for gaining a deeper understanding of the functionalities of the RhTPS and RhTPP gene families in rose.

18. Raymond, O.; Gouzy, J.; Just, J.; Badouin, H.; Verdenaud, M.; Lemainque, A.; Vergne, P.; Moja, S.; Choisne, N.; Pont, C.; et al. The Rosa genome provides new insights into the domestication of modern roses. Nature Genetics 2018, 50, 772-777, doi:10.1038/s41588-018-0110-3.

19.   Raza, A.; Bhardwaj, S.; Atikur Rahman, M.; García-Caparrós, P.; Habib, M.; Saeed, F.; Charagh, S.; Foyer, C.H.; Siddique, K.H.M.; Varshney, R.K. Trehalose: A sugar molecule involved in temperature stress management in plants. The Crop Journal 2023, doi:https://doi.org/10.1016/j.cj.2023.09.010.

20.   Fichtner, F.; Lunn, J.E. The Role of Trehalose 6-Phosphate (Tre6P) in Plant Metabolism and Development. Annu Rev Plant Biol 2021, 72, 737-760, doi:10.1146/annurev-arplant-050718-095929.

Comments 2:

No detail about the statistical analysis in M&M

Response 2:

We apologize for not providing detailed information on statistical analysis in the "Materials and Methods" section. To address this issue, we will make necessary revisions to the "Materials and Methods" section to provide more comprehensive details on statistical analysis. We will explicitly state the statistical methods employed, the analytical steps taken, and the relevant software used.

4.8 Data analysis

The data were analyzed using GraphPad Prism 9.0 (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com) in order to compare the differences between treatments. The figures display the mean ± standard deviation (SD) of biological triplicates, unless otherwise indicated. One-way analysis of variance was employed to determine statistical significance.

Correlation analysis was adopted using Pearson’s correlation. The package ggcorrplot was used for visualizations. p-Values less than 0.05 (p < 0.05) were displayed.

Comments 3:

The source of the plant material is not given.

Response 3:

Thank you for your comment. Indeed, the source of plant materials is crucial information in research to ensure the reproducibility and verifiability of experiments. We will clarify the source of materials and the mode of reproduction in the materials and methods.

4.1. Plant materials and treatments

In this study, Rosa hybrida cv ‘Carola’ was obtained from the Key Laboratory of Cold Region Landscape and Application, Northeast Agricultural University, Harbin, China (45.6811°N,126.6250°E). Using cuttage for propagation expansion.