Multi-Omics Analyses Uncover the Mechanism Underlying Polyploidization-Enhanced Steviol Glycosides Biosynthesis in Stevia rebaudiana

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The author employed the transcriptome, metabolome and microbiome to investigate the regulatory mechanism of SGs biosynthesis between autotetraploid and diploid stevia. The manuscript is clearly presented, with clear Tables and figures, and is recommended for acceptance with minor revisions.

Several points:

1) The current title does not bring out the full message of multi-omics, but over-emphasis the role of ‘microbe-plant interplay’ in regulating SGs biosynthesis. Please adjust it.

2) Line 29, gene name should be ‘5 NACs, 4 WRKYs, 3 MYBs, 8 bHLHs and 3 AP2/ERFs’.

3) Line 184, ‘UGT’ should be italic.

4) Figure 2D, Loss of indication information in image.

5) Figure 4, ‘D, diploid stevia rhizosphere; T, autotetraploid stevia rhizosphere’. Please standardize the abbreviated form.

6) Line 362, The abbreviation of 'Transcription factors' should be labelled when it first appear, and consistently throughout the manuscript, without alternating between full and abbreviated names.

7) Line 500, Subscript of ddH2O.

Author Response

Reviewer1

The author employed the transcriptome, metabolome and microbiome to investigate the regulatory mechanism of SGs biosynthesis between autotetraploid and diploid stevia. The manuscript is clearly presented, with clear Tables and figures, and is recommended for acceptance with minor revisions.

Dear Reviewers: Thanks for your comments and providing so many good suggestions on improving the performance of our manuscript. We have revised the manuscript according to your comments. The explanation to the comments point- by- point is as following.

Several points:

• The current title does not bring out the full message of multi-omics, but over-emphasis the role of ‘microbe-plant interplay’ in regulating SGs biosynthesis. Please adjust it.

REPLY：Thanks for your comments. The revised title is “Multi-omics analyses uncover the mechanism underlying polyploidization-enhanced steviol glycosides biosynthesis in Stevia rebaudiana”.

• Line 29, gene name should be ‘5 NACs, 4 WRKYs, 3 MYBs, 8 bHLHsand 3 AP2/ERFs’.

REPLY：Thanks for your suggestion. We have revised these genes format.

• Line 184, ‘UGT’ should be italic.

REPLY：We have revised this point.

• Figure 2D, Loss of indication information in image.

REPLY：Thanks for your comments. The indicative information of figured 2D has been completed.

• Figure 4, ‘D, diploid stevia rhizosphere; T, autotetraploid stevia rhizosphere’. Please standardize the abbreviated form.

REPLY：Thanks for your comments. We have standardized the abbreviation using ‘DSR, diploid stevia rhizosphere; TSR, autotetraploid stevia rhizosphere’ in Figure 4.

• Line 362, The abbreviation of 'Transcription factors' should be labelled when it first appears, and consistently throughout the manuscript, without alternating between full and abbreviated names.

REPLY：Thanks for your comments. we have revised this throughout the manuscript.

• Line 500, Subscript of ddH2O

REPLY：This point has been corrected.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The present manuscript corresponds to a revised version of a previous article submitted for publication in this Journal. I suggest its acceptance after minor revision as follows:

1. Please, revise the type of compounds in Table 1. For example, 4-hydroxy-3-methoxybenzoate is not a terpene.

2. CAS number is not necessary in Table 1.

3. Also in Table 1 - 2-Phenylethanol is not a phenolic acid. I suggest alteration for "phenolic compounds".

 

Author Response

Reviewer2

The present manuscript corresponds to a revised version of a previous article submitted for publication in this Journal. I suggest its acceptance after minor revision as follows:

1. Please, revise the type of compounds in Table 1. For example, 4-hydroxy-3-methoxybenzoate is not a terpene.

REPLY: Thanks for your reminder. In fact, this metabolite is 3β, 21β, 24-trihydroxyserrat-14-en-29-oic acid-3β (4-hydroxy-3-methoxybenzoate), which belongs to terpenoids. Sorry for the incorrect abbreviation.

2. CAS number is not necessary in Table 1.

REPLY: Thanks for your suggestion. We have deleted the CAS number in Table 1.

3. Also in Table 1 - 2-Phenylethanol is not a phenolic acid. I suggest alteration for "phenolic compounds".

REPLY: Thanks for your suggestion. We have moved 2-Phenylethanol the category ‘other’ to avoid ambiguity.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

The manuscript, entitled "Multi-omics analyses reveal microbe-plant interplay in enhancing steviol glycosides biosynthesis in Stevia rebaudiana post-autotetraploidization" (plants-3130443), submitted to the journal Plants, represents a study of the differences between diploid and autotetraploid stevia. The research employs metabolomic, RNA sequencing and ribosomal RNA amplicon sequencing approaches. The authors synthesise these studies to provide insights into the mechanisms underlying the increased production of metabolites of interest in varieties with a higher chromosome number.

It is a methodologically straightforward and neatly executed paper, well written, although the English language needs to be revised in detail in the Abstract (very confusing).

The article contains a considerable amount of text in red. I would be more amenable to this if it were a correction resulting from a prior arbitration. However, in this instance, I am encountering the article for the first time, and the file designation is "v1." If this is the initial version, there is no rationale for the text to be in a different color.

 

Other aspects that could be improved upon are listed below:

- Lines 36-37. Keywords are usually ordered alphabetically.

- Line 64. ‘harbors’. If the subject is The rizospheres the final s in harbors would be left out.

- Line 95. ‘and flavonoid content and antioxidant’. Please change to ‘, flavonoid content, and antioxidant’.

- line 137 and throughout most of the manuscript. There are many unnecessary capital letters (e.g. -Dihydroxy, -Tetrahydroxy in the line 138, Sphingolipids in the line 139, Triose-phosphate Transporter in the line 199, etc.). Please check the whole document (especially the tables).

- Line 140. LPE is not defined in the text.

- Line 142. ‘4 DAMs’. Below 10 is used in print. This also applies to the rest of the document (line 221, for example).

- Lines 156, 161 and the whole document. stevia, in lower case, is what they use in almost the whole document, it is the common name of the plant, and it is correct. But Stevia, capitalised, would be the scientific name of the genus of the plant and in that case, it should be written in italics. Please be consistent in your wording.

- Lines 384-385. Bacillus should be written in italics.

- Line 398. Basidiomycota, on the other hand, should be written without italics.

- Line 439. 5 ml. You use the terminology mL throughout the document, be homogeneous.

- Line 441. 2500 g. Please, change to 2500 × g. g is written in italics.

- Lines 445-514. The spacing of these paragraphs is different from the rest of the document.

- Line 478. ‘2.1 mm * 100 mm)’. Please, change to 2.1 mm × 100 mm).

-Line 500. ddH2O. Please, change to ddH₂O.

Comments on the Quality of English Language

English language needs to be revised in detail in the Abstract (very confusing).

Author Response

Reviewer3

The manuscript, entitled "Multi-omics analyses reveal microbe-plant interplay in enhancing steviol glycosides biosynthesis in Stevia rebaudiana post-autotetraploidization" (plants-3130443), submitted to the journal Plants, represents a study of the differences between diploid and autotetraploid stevia. The research employs metabolomic, RNA sequencing and ribosomal RNA amplicon sequencing approaches. The authors synthesise these studies to provide insights into the mechanisms underlying the increased production of metabolites of interest in varieties with a higher chromosome number.

It is a methodologically straightforward and neatly executed paper, well written, although the English language needs to be revised in detail in the Abstract (very confusing).

The article contains a considerable amount of text in red. I would be more amenable to this if it were a correction resulting from a prior arbitration. However, in this instance, I am encountering the article for the first time, and the file designation is "v1." If this is the initial version, there is no rationale for the text to be in a different color.

REPLY: Thanks for your comments. We have revised the abstract to make it more cleared. The red markings in the text indicate revisions made following the suggestions of the two previous reviewers, as well as adjustments based on the editor's comments prior to resubmission. We apologize for the file version being labeled as 'V1'.

Other aspects that could be improved upon are listed below:

- Lines 36-37. Keywords are usually ordered alphabetically.

- Line 64. ‘harbors’. If the subject is the rizospheres, the final s in harbors would be left out.

- Line 95. ‘and flavonoid content and antioxidant’. Please change to ‘, flavonoid content, and antioxidant’.

- line 137 and throughout most of the manuscript. There are many unnecessary capital letters (e.g. -Dihydroxy, -Tetrahydroxy in the line 138, Sphingolipids in the line 139, Triose-phosphate Transporter in the line 199, etc.). Please check the whole document (especially the tables).

- Line 140. LPE is not defined in the text.

- Line 142. ‘4 DAMs’. Below 10 is used in print. This also applies to the rest of the document (line 221, for example).

- Lines 156, 161 and the whole document. stevia, in lower case, is what they use in almost the whole document, it is the common name of the plant, and it is correct. But Stevia, capitalised, would be the scientific name of the genus of the plant and in that case, it should be written in italics. Please be consistent in your wording.

- Lines 384-385. Bacillus should be written in italics.

- Line 398. Basidiomycota, on the other hand, should be written without italics.

- Line 439. 5 ml. You use the terminology mL throughout the document, be homogeneous.

- Line 441. 2500 g. Please, change to 2500 × g. g is written in italics.

- Lines 445-514. The spacing of these paragraphs is different from the rest of the document.

- Line 478. ‘2.1 mm * 100 mm)’. Please, change to 2.1 mm × 100 mm).

-Line 500. ddH2O. Please, change to ddH₂O.

REPLY: Thank you for providing constructive comments to improve the manuscript. We have revised the manuscript according to your comments and marked all changes in the revised manuscript.

Reviewer 4 Report (New Reviewer)

Comments and Suggestions for Authors

Manuscript titled “Multi-omics analyses reveal microbe-plant interplay in enhancing steviol glycosides biosynthesis in Stevia rebaudiana post-autotetraploidization” reports various analyses aimed at determining the differences between diploid and tetraploid stevia plants. The authors used sensitive and thorough analyses to verify differences in metabolites, gene expression, microbial populations and others, and integrated them into a comprehensive work. The work is interesting and well-written. There are some comments and suggestions for the authors:

 

1.       In line 55, should “ent-kaurenoic acid (ent-KAH)” be “ent-kaurenoic acid hydrolase (ent-KAH)” instead?

2.       The introduction highlights the commercial value of stevia plants for their sweet flavor. Is it known if plants grown for this purpose are diploid or tetraploid? Or is this aspect not considered (or at least reported) for commercial plants?

3.       In Table 1, please consider using “retinol” and “riboflavin” instead of vitamins A1 and B2, respectively. This may be more informative and better coincides with the other molecules, whose names are written in full.

4.       Please check Figure 2D, since it appears that some text from the legend has been cropped on the right.

5.       In Figure 3:

a.       “Red and green indicate upregulated and downregulated genes, respectively”. Is it possible to change downregulated genes from green to blue? This would better coincide with Figure 3B, which uses red to indicate positive changes and blue to indicate negative ones.

b.       What does the dashed arrow indicate, as compared to the solid ones? Please specify in the legend.

6.       Lines 335-337 highlight that higher SGs in tetraploid plants suggest they have potential applications in the food industry. Although likely true, this hypothesis requires additional experimental evidence to fully validate. Perhaps the need for such experiments could be mentioned for future works.

Author Response

Reviewer4

Manuscript titled “Multi-omics analyses reveal microbe-plant interplay in enhancing steviol glycosides biosynthesis in Stevia rebaudiana post-autotetraploidization” reports various analyses aimed at determining the differences between diploid and tetraploid stevia plants. The authors used sensitive and thorough analyses to verify differences in metabolites, gene expression, microbial populations and others, and integrated them into a comprehensive work. The work is interesting and well-written. There are some comments and suggestions for the authors:

1. In line 55, should “ent-kaurenoic acid (ent-KAH)” be “ent-kaurenoic acid hydrolase (ent-KAH)” instead?

REPLY: Thanks for your suggestion. We have modified this point.

2. The introduction highlights the commercial value of stevia plants for their sweet flavor. Is it known if plants grown for this purpose are diploid or tetraploid? Or is this aspect not considered (or at least reported) for commercial plants?

REPLY: Thank you for your question. Currently, diploid stevia is mainly grown on the market and is widely used in industry. The test material used in this study was tetraploid stevia obtained through colchicine induction in our laboratory and has not yet been promoted on the market. At present, this study found that tetraploid stevia has a higher content of active ingredients and superior phenotype. In addition, the expression level of genes that regulate the synthesis of the key metabolite steviol glycosides and the content of beneficial microorganisms in autotetraploid stevia has significantly improved in comparison with diploid stevia. Therefore, based on preliminary research, we believe that tetraploid stevia has more potential commercial value than diploid stevia.

3. In Table 1, please consider using “retinol” and “riboflavin” instead of vitamins A1 and B2, respectively. This may be more informative and better coincides with the other molecules, whose names are written in full.

REPLY: Thanks for your valuable comments. We have revised these two compounds’ name.

4. Please check Figure 2D, since it appears that some text from the legend has been cropped on the right.

REPLY: Thanks for your comments. The indicative information in figured 2D has been completed.

5. In Figure 3: a)“Red and green indicate upregulated and downregulated genes, respectively”. Is it possible to change downregulated genes from green to blue? This would better coincide with Figure 3B, which uses red to indicate positive changes and blue to indicate negative ones. b) What does the dashed arrow indicate, as compared to the solid ones? Please specify in the legend.

REPLY: Thanks for your suggestions.

a) We have modified figure 3B using blue to indicate downregulated gene.

b) We have changed the dashed arrow to a solid arrow.

6. Lines 335-337 highlight that higher SGs in tetraploid plants suggest they have potential applications in the food industry. Although likely true, this hypothesis requires additional experimental evidence to fully validate. Perhaps the need for such experiments could be mentioned for future works.

REPLY: Thanks for your comments. Based on the superior characteristics of tetraploid plants compared to diploid plants previously reported, our laboratory used colchicine to prepare tetraploid stevia. This study preliminarily explored the advantages of tetraploid stevia at the genetic, metabolite and microbial levels compared with diploid stevia. Subsequent field trials are also planned to evaluate the quality and yield of the tetraploid stevia variety, using diploid stevia as a reference to further verify its economic value and application potential. In the future, new varieties of tetraploid stevia will be considered for marketing. And we have stated this in the Discussion section.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is plenty of statistics and probabilities, but real scientific evidence is rather lacking. The authors “identified” 916 differentially accumulated metabolites influenced by polyploidisation. The authors should give more information on how all these compounds were separated and quantified with a single Agilent SB-C18 column (calibration curves, further identification by NMR, SD…). Quantitation of steviol glycosides is very difficult as shown by round-robin testing performed by specialised laboratories even of large companies world-wide. This was done by using external and internal standard methods. Even with standard mixtures of ultra-pure compounds, the analyses by some laboratories were often unacceptable. Therefore, as the steviol glycoside content is of utmost importance in this kind of studies, more information should be given, e.g., calibration curves, purity of standards, SD etc.

 2) Most of the literature cited is of the last 5 years and no reference is made to the original research.

 3) Anti-diabetic effects of steviol glycosides are claimed without further discussion. Assays with rats and mice indeed suggest possible effects in man, but most of the recent studies with human volunteers, performed at different research locations, could not confirm the claimed anti-diabetic effects of steviol glycosides.

 4) Plant material:

The tetraploid plants were obtained after colchicine treatment. Can they then be called “auto”-tetraploid? Three biological replicates were taken within 1 experiment. Will similar results of the 916 compounds be obtained when the experiment is repeated 2 or 3 times?

5) The literature on rhizosphere microbiomes describes possible effects in different plant families often not related with Stevia (e.g., for nitrogen fixation). The authors should focus more on the literature concerning stevia and related plants

Comments on the Quality of English Language

e.g.: line 98-99: sentence is not complete

238: DSL "represents" ?

241: .. the different (of) omit "of" rhizosphere

267: The dots represent a...

440: for each ploidy type.

476: The mobile phase was consisted... omit "was"

486: were further optimized.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Multi-omics analyses reveal microbe-plant interplay in enhancing steviol glycosides biosynthesis in Stevia rebaudiana post-autotetraploidization” is interesting, well-structured and explained in accessible language.  The subject matter and layout of the manuscript meet the requirements of the journal Plants.

 

Most of the publications cited are from the last 5 years, which shows the innovation and relevance of the chosen research topic.

I suggest the following minor corrections:

Line 15 - As this is the first mention of stevia in your manuscript, please add the full botanical name along with the author who identified the species - Stevia rebaudiana (Bertoni)

Line 36 - Please choose keywords that do not repeat words from the title. I recommend removing Stevia rebaudiana and steviol glycosides, and adding UDP-glycosyltransferases, for example.

Line 59 – enzyme abbreviations (UGT73E1, UGT74G1, UGT76G1, UGT85C2 and UGT91D2) should not be italicized

Line 66 – “associated” not “associations”

Line 99 – The sentence is not correct: “further investigation” is redundant or, if it remains, should be added "and need” further investigation.

Line 141, 142 – "in higher amount" should be added: ".... were accumulated in higher amount in autotetraploid ....." Otherwise, these four DAMs appear to accumulate only in tetraploid stevia.

Line 196 – “….. three sugar efflux transporter” – please add "genes" to avoid confusion with proteins.

Line 200 - The designation UGNT1 occurs in two more places in the publication, but with the addition _ARATH. Perhaps it should be added here as well.

Line 226 - ent-KAH and ent-KS1 should be italicized when denoting genes.

Line 289 – gene abbreviations should be in italic

Line 360 - regulating the expression of other structural genes - "of" should be added

Line 368 – "families" not "family"

Line 376 – In my opinion, “provide” is more appropriate than “yield”

Line 387 – “such as” is more appropriate than “like”

Line 436 – Transcriptomic to be with lowercase

Line 486 – “optimized” not “optimization”

Lines 542, 543 – the phrase “molecular and physiological metabolism” is incorrect. Only “metabolism” is more appropriate.

Lines 676, 691, 726 – 35., 42. and 58. Except for the first word in the title, the rest words should begin with a lowercase letter.

Line 678 – 36. The year of publication of the article is missing – 2023; “208” not bold.

Line 705 – The name Bertoni should be capitalized.

Comments for author File: Comments.pdf