Review Reports - Transcriptome-Wide Identification of miRNAs and Their Targets During Riboflavin-Promoted Dormancy Release in <i>Lilium</i> ‘Siberia’

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I have a few concerns to be addressed in the respective part of this manuscript. Please see below and address them accordingly.

1. line 15, rempve space before 'y'

2. line 162, correct 'oC'

3. line 165, correct H2O2 formula

4. Line 238-248, please write in a paragraph

5. line 549, correct figure 'A'

6. In introduction, please mention reason of selecting L. Oriental instead of L. longiflorum

7. Check your citation and journal style. need to follow the style

8. Why 0.1 mM riboflavin showed more effective than 0.5 mM for promoting dormancy release and flowering in L. Oriental? Was there any hypothesis or prior research guiding these concentration choices? if so, need the citations.

9. How miRNAs (like miR395-y and miR529-z) worked in the dormancy release mechanism?

10. The manuscript mentions different mechanisms that influenced by riboflavin, such as oxidative phosphorylation and flavonoid metabolism. Please explain how these pathways interact.

11. In this ms more upregulated than downregulated genes after riboflavin treatments in gene expression. Could you discuss the functional significance of this imbalance and how it might reflect on dormancy release mechanisms?

Please make your words concise. I am looking forward to see the revised file.

Comments on the Quality of English Language

Author Response

I have a few concerns to be addressed in the respective part of this manuscript. Please see below and address them accordingly.

line 15, rempve space before 'y'

The author’ answer: Thank you for your comment. We have modified this in the manuscript in line15.

line 162, correct 'oC'

The author’ answer: Thank you for your comment. We have modified this in the manuscript in line166.

line 165, correct H2O2 formula

The author’ answer: Thank you for your comment. We have checked and used the correct formula of H₂O₂ in the whole manuscript.

Line 238-248, please write in a paragraph

The author’ answer: Thank you for your comment. We have modified and writed that in a paragraph.

line 549, correct figure 'A'

The author’ answer: Thank you for your comment. We have modified it into Figure 5'C' in line 540.

In introduction, please mention reason of selecting L. Oriental instead of L. longiflorum

The author’ answer: Thank you for your comment. There are significant differences in their dormancy characteristics, between L. Oriental and L. longiflorum. L. longiflorum has a short growth period, flowering within one year of sowing, tolerates high temperatures, but is more sensitive to light; Whereas the L. Oriental bulb is sensitive to temperature, intolerant of heat, and enters dormancy during summer's high temperatures to maintain normal growth and flowering. Therefore, the L. Oriental is more suitable material for studying the impact of dormancy characteristics on the quality of lily cut flowers. We have supplemented in line50-line55.

Check your citation and journal style. need to follow the style

The author’ answer: Thank you for your comment.We have modified it in references citation.

Why 0.1 mM riboflavin showed more effective than 0.5 mM for promoting dormancy release and flowering in L. Oriental? Was there any hypothesis or prior research guiding these concentration choices? if so, need the citations.

The author’ answer: Thank you for your comment. Previous studies have not reported the effect of riboflavin in the process of dormancy release of lily bulb, but 0.5 mM riboflavin can promote the differentiation of globular embryo(GE) and size of GE are larger than control during longan somatic embryogenesis (Xu et al., 2023). In our study, 0.5 mM riboflavin concentration may be too high for lily bulb dormancy, and the promotion effect is not as good as that of 0.1 mM riboflavin. It is possible that riboflavin has different concentration ranges for different growth and development processes of different plants. We have cited it in the manuscript on line690-693, line786.

[1] Xu, X.P., Zhang, C.Y., Xu, X.Q., Cai, R.D., Guan, Q.X., Chen, X.H., Chen, Y.K., Zhang, Z.H., XuHan, X., Lin, Y.L., Lai, Z.X., 2023a. Riboflavin mediates m6A modification targeted by miR408, promoting early somatic embryogenesis in longan. Plant Physiol. 192, 1799-1820. https://doi.org/10.1093/plphys/kiad139.

How miRNAs (like miR395-y and miR529-z) worked in the dormancy release mechanism?

The author’ answer: Thank you for your comment. In 3.4(on line464), we can understand that miR-395-y was predicted to target APS1, APS1 participates sulfur metabolism, this pathway was significant enrichment in 0.1 mM riboflavin treatment group, and may participate in promoting dormancy release; miR529-z were predicted to target SPL14, as previous research showed that AtSPL14 is a negative regulator of vegetative-phase changes and floral transitions[1,2], Furthermore, in O. sativa, higher expression of OsSPL14 can reduce the tiller number, increase the lodging resistance, promote panicle branching and enhance grain yields[3,4]. Similarly, it is predicted that other differentially expressed miRNAs (miR396-y, miR399-y, miR164-y, miR168-x, miR1863-z) may target genes(CFDP1, DIT1, TRAF1A, AGO1B) and participate in the growth and development of bulbs and the primary metabolism pathways,then regulating the dormancy release of bulbs.

[1]Stone, J.M.; Liang, X.; Nekl, E.R.; Stiers, J.J. Arabidopsis AtSPL14, a plant-specific SBP-domain transcription factor, participates in plant development and sensitivity to fumonisin B1. Plant J. 2005, 41, 744–754.

[2]Lei M, Li ZY, Wang JB, Fu YL, Ao MF, Xu L. Constitutive Expression of Aechmea fasciata SPL14 (AfSPL14) Accelerates Flowering and Changes the Plant Architecture in Arabidopsis. Int J Mol Sci. 2018 Jul 18;19(7):2085. doi: 10.3390/ijms19072085.

[3]Jiao, Y.; Wang, Y.; Xue, D.; Wang, J.; Yan, M.; Liu, G.; Dong, G.; Zeng, D.; Lu, Z.; Zhu, X.; et al. Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nat. Genet. 2010, 42, 541–544.

[4]Miura, K.; Ikeda, M.; Matsubara, A.; Song, X.J.; Ito, M.; Asano, K.; Matsuoka, M.; Kitano, H.; Ashikari, M. OsSPL14 promotes panicle branching and higher grain productivity in rice. Nat. Genet. 2010, 42, 545–549.

The manuscript mentions different mechanisms that influenced by riboflavin, such as oxidative phosphorylation and flavonoid metabolism. Please explain how these pathways interact.

The author’ answer: Thank you for your comment. In 3.5 (on line 513-540). First, the transcriptional abundance of LoPAP17 in the apical buds of Lilium 'Siberia' bulbs was significantly upregulated after treatment with different concentrations of riboflavin (Figure 5.A), which might further active an increase in the endogenous riboflavin content (Figure 5.B, C) and affect the FMN levels (Figure 5.A). Next, riboflavin is the precursor of FMN and FAD, FMN and FAD participate in the assembly of complex I and complex II in the oxidative phosphorylation process on the inner mitochondrial membrane, and NAD+ and H+ are produced. The upregulated expression of F-type ATPase in complex V activated the oxidative phosphorylation pathway to produce ATP and H+ (Figure 5.A). The energy produced is involved in carbohydrate metabolism, glycolysis metabolism, amino acid metabolism, fatty acid metabolism, photosynthetic system reactions, and flavonoid metabolism in the cytoplasm.

In this ms more upregulated than downregulated genes after riboflavin treatments in gene expression. Could you discuss the functional significance of this imbalance and how it might reflect on dormancy release mechanisms?

The author’ answer: Thank you for your comment. According to the annotation of differential expressed (DE) genes, more upregulated genes after riboflavin treatment are primarily annotated in photosynthetic system energy metabolism (LHCA4, CAB40, CAB13, RBCS3), and blue light receptor phototropin synthesis gene (PHOT2); Starch and sucrose metabolism (endoglucanase 6), fructose and mannose metabolism (MAN1), and dihydrofolate reductase (FSH3); Cysteine and methionine metabolism (MAO1B, 1-aminocyclopropane-1-carboxylic oxidase), zinc ion binding (FLZ5) proteins, riboflavin metabolism (PAP17), glutathione metabolism (GSTF12); Plant hormone signal transduction (ERF.C.3), flavonoid and flavonol metabolism (CHS5, CHS1, RT), flavonoid compound biosynthesis (ANT18), anthocyanin synthesis (ANS), acyltransferase activity gene (acetyltransferase, SHT), fatty acid biosynthesis (CHS5, CHS1, C2, GDSL esterase/lipase, EXL3), lipid metabolism (LOX1.5, LPXD1, LTP203), glycine, serine, and threonine metabolism (HTH). These pathways are predominantly primary metabolism, with secondary metabolism mainly involving flavonoid metabolism, fatty acid metabolism, and lipid metabolism. It was suggested that riboflavin activated photosynthetic system energy metabolism, starch and sucrose metabolism, fructose and mannose metabolism, folate metabolism, and riboflavin metabolism et al. The activation of these metabolic pathways provides the necessary substances and energy for redox reactions, RNA and DNA synthesis, protein synthesis, and the differentiation and growth of apical meristem cells involved in the process of bulb dormancy release[1,2]. Glutathione metabolism not only participated photosynthesis but also regulated precursor to synthetic plant hormones such as ethylene synthesis and auxin synthesis, ultimately influences the release of plant dormancy.‌ Fatty acid metabolism generates acetyl-CoA, which influences abscisic acid (ABA) synthesis through the citric acid cycle pathway.

However, downregulated genes were mainly annotated as SULTR1;3, 5'-adenylylsulfate reductase 3, APS1 and SDI1 during dormancy release after 0.1 mM riboflavin treatment, which mainly participated sulfur metabolism. Arabidopsis seed germination experiments indicate a reduction in intracellular sulfate levels, leading to a decrease in cysteine precursor synthesis and a significant reduction in ABA content within the cells[3]. Previous reports indicated that reduced ABA synthesis promotes dormancy release[4]. SPL14 was downregulated and targeted by miR529-z. As previous research showed that AtSPL14 is a negative regulator of vegetative-phase changes and floral transitions[5,6]. The release of dormancy in bulbous plants is also the result in a large count of gene differential expression. Multiple primary and secondary metabolic pathways interact and regulate each other, ultimately affecting dormancy release.

We have also supplemented the discussion in line761-line770.

[1]Zhao Y, Liu C, Sui J, Liang J, Ge J, et al. A wake-up call: signaling in regulating ornamental geophytes dormancy. Ornamental Plant Research. 2022. 2:8 doi: 10.48130/OPR-2022-0008.

[2]Zhu H, Chen Z, Zhang K, Cui J. The key role of sugar metabolism in the dormancy release and bud development of Lilium brownii var. viridulum Baker bulbs. Scientia Horticulturae, 2024. 336: 113453.

[3]Chen Z, Zhao PX, Miao ZQ, Qi GF, Wang Z, Yuan Y, Ahmad N, Cao MJ, Hell R, Wirtz M, Xiang CB. SULTR3s Function in Chloroplast Sulfate Uptake and Affect ABA Biosynthesis and the Stress Response. Plant Physiol. 2019 May;180(1):593-604. doi: 10.1104/pp.18.01439.

[4]Wu J, Jin Y, Liu C, Vonapartis E, Liang J, Wu W, Gazzarrini S, He J, Yi M. GhNAC83 inhibits corm dormancy release by regulating ABA signaling and cytokinin biosynthesis in Gladiolus hybridus. J Exp Bot. 2019 Feb 20;70(4):1221-1237.

[5]Stone, J.M.; Liang, X.; Nekl, E.R.; Stiers, J.J. Arabidopsis AtSPL14, a plant-specific SBP-domain transcription factor, participates in plant development and sensitivity to fumonisin B1. Plant J. 2005, 41, 744–754.

[6]Lei M, Li ZY, Wang JB, Fu YL, Ao MF, Xu L. Constitutive Expression of Aechmea fasciata SPL14 (AfSPL14) Accelerates Flowering and Changes the Plant Architecture in Arabidopsis. Int J Mol Sci. 2018 Jul 18;19(7):2085. doi: 10.3390/ijms19072085.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

After reading the manuscript entitled “Transcriptome-wide identification of miRNAs and their targets during riboflavin-promoted dormancy release in Lilium Orient- al hybrid ‘Siberia’’” by Xiaoping Xu , Chenglong Yang , Yiping Zheng , Wenjie Guo , Zhongxiong Lai * , Shaozhong Fang *. I recognize the content of this manuscript as robust and scientifically up-to-date in terms of the techniques used.

I have a few suggestions and I hope the authors will think about them. One of them is the key words used, replace them as they are all in the title. The introduction is too long. I've noticed that the last paragraph could be removed and the hypothesis and research objectives used instead. I see that the authors' conclusions are very long, so I ask: did it meet the proposed objectives? I think it would be worth evaluating this question.

item 2.2 should be more detailed. Some information appears in the results and should be clear in the material and methods.

The return on the research should be clear in the text, I ask: what is the return on your research and publication for the scientific and non-scientific community?

Author Response

One of them is the key words used, replace them as they are all in the title.

The author’ answer: Thank you for your comment. We have modified the title into “Transcriptome-wide identification of miRNAs and their targets during riboflavin-promoted dormancy release in Lilium ‘Siberia’ ”.

The introduction is too long. I've noticed that the last paragraph could be removed and the hypothesis and research objectives used instead.

The author’ answer: Thank you for your comment. We have modified the last paragraph in the introduction, in line112-line121.

I see that the authors' conclusions are very long, so I ask: did it meet the proposed objectives? I think it would be worth evaluating this question.

The author’ answer: Thank you for your comment. We have modified the conclusion in line829-line845.

item 2.2 should be more detailed. Some information appears in the results and should be clear in the material and methods.

The author’ answer: Thank you for your comment.We have modified the item 2.2 in line150, and clear it in the results in line322.

The return on the research should be clear in the text, I ask: what is the return on your research and publication for the scientific and non-scientific community?

The author’ answer: Thank you for your comment. For the scientific, our findings provide new insights into the potential regulatory mechanisms of miRNAs and their targets respond to riboflavin and promote bulb plant dormancy release for the first time. The study suggests that riboflavin may participate in the molecular mechanisms of plant dormancy release through participating in epigenetic regulation, filling the blank of the biological function of riboflavin in plant development specially in the dormancy release process of bulbous plants. For non-scientific community, our research provides a feasible basis for shortening the dormancy-release time of lilies and promoting their flowering through artificial application of riboflavin in the field.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The paper is missing figures and supplementary. Overall there are many concerns, and without its clarification the work cannot be evaluated.

The project mentioned in the paper (PRJNA1179346) is very suspicious - no abstract, no authors, no description, many broken links.

“mRNA transcriptome databases of apical buds treated with different concentrations of riboflavin“ - this cannot be true in principle, 2 datasets in NCBI and three treatments here.

Opposite effect of Riboflavin on H2O2 after 30d and 60d storage. Fig 2EF

No supplementary figures can be found.

Variation in expression within one type of samples is huge, obvious replication problems Fig 3 CD. I may expect that the most pronounced effect should be right after the treatment, so 30d should be more different from Mock as 60d. This is not the case on fig 3. More, genes mir164 mir395 have similar expression in two experiments, how they can be detected as DE?

Fig 4 - prediction of miRNA targets has high false positive rate, presumably all of the predicted is falls. is there any correlation between miRNA and target genes in expression levels. Authors should give supp table with data.

Fig 5 A is missing

How endogenous riboflavin content was measured on fig 5B, and contrasted to exogenous riboflavin?

Fig 6 supports the false prediction of miRNA targets, many pairs miRNA-target genes have unexplainable expression, best example - loDIT1.

IT is not clear why localization in the nucleus is important for dormancy release or riboflavin production - fig 8

Author Response

1.The paper is missing figures and supplementary. Overall there are many concerns, and without its clarification the work cannot be evaluated.

The author’ answer: Thank you for your comment. We have uploaded our supplementary file in the system, including Figures and Tables.

2.The project mentioned in the paper (PRJNA1179346) is very suspicious - no abstract, no authors, no description, many broken links.

The author’ answer: Thank you for your comment. PRJNA1179346 is the accession number of the full-length transcriptome of Lilium ' Siberia '. The submission number of SRA is SUB14822704, the title is “The full length transcriptome of Lilium Oriental hybrid Siberia”; authors and submitter is Xiaoping Xu, E-mail: byxxp310107@163.com; description is “A third-generation full-length transcriptome database was constructed for comparison of lily miRNA and mRNA databases”. The links “https://submit.ncbi.nlm.nih.gov/subs/sra/SUB14822704/overview”.

“mRNA transcriptome databases of apical buds treated with different concentrations of riboflavin“ - this cannot be true in principle, 2 datasets in NCBI and three treatments here.

The author’ answer: Thank you for your comment. The Bioproject number of mRNA transcriptome databases is PRJNA1063590. The Links is “ https://dataview.ncbi.nlm.nih.gov/object/PRJNA1063590 ”. There are three datasets in three treatments respectively, the name is Mock-1, Mock-2, Mock-3; R1-1, R1-2, R1-3; R2-1, R2-2, R2-3.

Opposite effect of Riboflavin on H2O2 after 30d and 60d storage. Fig 2EF

The author’ answer: Thank you for your comment. At 30 d, the Mock has not broken dormancy, while different concentration riboflavin treatment have broken dormancy, the level of H₂O₂ also higher than Mock. At 60 d, the Mock also broke dormancy, the absolute value of H₂O₂ was higher than that at 30 d, and the lily bulbs treated with different concentrations of riboflavin were in the late stage of dormancy release, and the H₂O₂ content decreased at this time. As previously reported, H₂O₂ levels gradually increase during dormancy maintenance to dormancy release, reaching their peak when dormancy is fully release, and then gradually decrease during the later stages of dormancy release. This also affects endogenous ABA and GA₃ levels[1], dynamically regulating the physiological state of bulb dormancy release.

[1]Liu Y, Ye N, Liu R, Chen M, Zhang J. H2O2 mediates the regulation of ABA catabolism and GA biosynthesis in Arabidopsis seed dormancy and germination. J Exp Bot. 2010 Jun;61(11):2979-90.

No supplementary figures can be found. Variation in expression within one type of samples is huge, obvious replication problems Fig 3 CD. I may expect that the most pronounced effect should be right after the treatment, so 30d should be more different from Mock as 60 This is not the case on fig 3. More, genes mir164 mir395 have similar expression in two experiments, how they can be detected as DE?

The author’ answer: Thank you for your comment. We have uploaded our supplementary files in the system. Fig 3C represents TPM values of DE-known miRNAs in three replicates of three treatment group, Fig 3D represents TPM values of novel miRNAs in three replicates of three treatment group. Please see the specific data in TableS3. For the study of lily bulb dormancy release, we pay more attention to the changes of endogenous miRNA expression during the transition stage from dormancy maintenance to dormancy release after riboflavin treatment, so we used the materials after different concentration riboflavin treated 30d to analyze the miRNA network. From the TPM of miR164 and miR395, compared with Mock, they just have the same expression pattern, and downregulated during different groups (Mock, R1, R2), moreover they all differential expression(DE) compare with Mock group.

Fig 4 - prediction of miRNA targets has high false positive rate, presumably all of the predicted is falls. is there any correlation between miRNA and target genes in expression levels. Authors should give supp table with data.

The author’ answer: Thank you for your comment. The correlation of miRNA and target genes have been verified in expression levels by qRT-PCR, please see supplementary file TableS8.

Fig 5 A is missing

The author’ answer: Thank you for your comment. We have added it in the manuscript.

How endogenous riboflavin content was measured on fig 5B, and contrasted to exogenous riboflavin?

The author’ answer: Thank you for your comment. In line175, we described the determination of riboflavin content, HPLC was used to detect the riboflavin content. In Fig5B, the horizontal axis represents the different concentration of exogenous riboflavin treatment, the Y-axis represents the endogenous riboflavin, the content of endogenous riboflavin increased with the increase of exogenous riboflavin treatment.

Fig 6 supports the false prediction of miRNA targets, many pairs miRNA-target genes have unexplainable expression, best example - loDIT1.

The author’ answer: Thank you for your comment. The negative relationship of miRNA and targets may also be influenced by the external different treatment, the same target gene may be regulated by multiple miRNAs, the regulation of target genes by miRNA is complex and diverse. They may exhibit typical negative regulatory relationships under all different treatments, but they might display a typical negative regulatory model at a specific stage. In TableS7, we obtain all miRNA predicted targets. After we use qRT-PCR to verified the relationship of the miRNA and their targets, from the result in Fig6, they are still some miRNA and their target have negative relationship in all different treatment groups, such as miR396-y and LoCFDP1, miR1863-z and LoFBA3. As for other miRNA, such as miR529-z and LoSPL14, they may be just have negative relationship in some groups, such as 0.1 mM and 0.5 mM riboflavin treatment. MiR399-y and LoDIT1, they may be just have negative relationship in 0.1 mM riboflavin. MiR395-y was significantly upregulated after 0.5 mM riboflavin treatment, while the target gene LoAPS1 was significantly downregulated.

IT is not clear why localization in the nucleus is important for dormancy release or riboflavin production - fig 8

The author’ answer: Thank you for your comment. Our data demonstrated that exogenous riboflavin treatment directly upregulated the expression of LoPAP17, LoPAP17 participated in riboflavin metabolism, and promoted riboflavin and FMN accumulation. Moreover, LoPAP17 is located in the nucleus (Figure 8.). SignalP-6.0 prediction showed that LoPAP17 protein was predicted as a signal peptide (Sec/SPI), and cleavage site between pos. 26 and 27, probability is 0.973517 (FigS5E, Fig A). TMHMM2.0 prediction showed that LoPAP17 has the possibility of being a transmembrane protein by 0.52556 (Fig B). These characteristics suggest that LoPAP17 may have abundant biological functions, this suggested us whether LoPAP17 has other molecular functions beyond promoting riboflavin biosynthesis, which we will explore further in subsequent projects. Simultaneously, in mitochondria, FMN and FAD participate in the assembly of complex I and complex II and upregulate the F-type ATPase of complex V in the oxidative phosphorylation process. The energy produced was involved in carbohydrate metabolism, glycolysis metabolism, amino acid metabolism, sulfur metabolism, fatty acid metabolism and photosynthetic system reactions, they jointly regulated the process of dormancy release in lily bulbs.

Fig. SignalP-6.0 and TMHMM2.0 prediction of LoPAP17 protein. A represents SignalP-6.0 prediction of LoPAP17, B represents the TMHMM2.0 prediction of LoPAP17.