Functional Analysis of Glucose-6-Phosphate Translocator PoGPT1 in Embryogenic Callus Formation of Tree Peony

Round 1

Reviewer 1 Report (New Reviewer)

Dear Authors,

 I offer the following comments to the manuscript:

1)      The authors focus much of the introduction and discussion on the process of somatic embryogenesis. And they often interpret the regulatory pathways involved in this process on the basis of the results obtained here without documenting somatic embryogenesis in this paper (obtaining embryogenic callus does not prove that regeneration by somatic embryogenesis occurs!!!) Hence, the question is whether the authors are able to document the formation of somatic embryos from transgenic callus? If not such conclusion should be omitted and clearly written that it is  only speculation.

2)      If somatic embryogenesis can be induced in this spices why Authors didn’t use somatic embryo to expression analysis of GPT1?

3)      As the authors conducted studies on different fragments of the plant, zygotic embryos and callus please clearly specify the purpose of the presented work.

4)      Please explain all abbreviations when used for the first time.

5)      Please make it clear when you are writing about zygotic versus somatic embryogenesis, and somatic embryogenesis versus other in vitro regeneration processes. In many places in the work, the reader loses clarity about what the particular fragment is about.

6)      Given the results obtained, the introduction should not place so much emphasis on improving the efficiency of somatic embryogenesis and conversion of somatic embryos because this was ultimately not demonstrated in this study.

 TITLE

The title is inadequate to the overall work and omits completely: functional analysis of GPT1, its localization in the plant and expression in zygotic embryo, and expression of transcription factors in calli. So  4 of the 6 points of the results were not included in the title. Moreover, the use of the phrase "formation of embryogenic cells" in the title and not showing the ultrastructure of embryogenic and non-embryogenic cells is unjustified. That's why I propose to change the title and bring it in line with all aspects undertaken in the work.

ABSTRACT

Line 21: Throughout the manuscript, there is no information on how the development of embryos in Peonia ostia proceeds. Also absent is any photographic documentation showing the embryos. On what basis do the authors conclude that PoGPT1 transcription increases during embryo development????

Line 26-28: As the authors do not document in the paper the regeneration of somatic embryos they obtained, or the efficiency of somatic embryogenesis (only embryogenic callus is presented) this sentence does not hold true.

INTRODUCTION

Line 49-50: Please explain the difference between vegetative and somatic. From my point of view means the same.

RESULT

My biggest objection in this part is to the lack of visualization of the statistical analysis in the graphs included in the Results. Please supplement the graphs with information on which means are different from each other. Without this the results cannot be interpreted!

  REFERENCES

This section should be examined again In terms of italics for species and gene names (some marked in the pdf).

 25 and 44 are the same publication!!!

 FIGURES

Figures are not very precisely described: there are no information on day of culture, the titles of the figures often do not refer to all the pictures on them.

 All figures on which quantitative analyses are presented (Figs. 5, 7, 8) , do not contain statistics only deviations are marked. No information on which samples are statistically different from each other. Please mark the differences. Without this, the results cannot be interpreted and all conclusions drawn are unjustified.

  Respectfully, Reviewer

 Comments for author File: Comments.pdf

Author Response

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Reviewer 2 Report (New Reviewer)

1.    How was it proven that PoGPT1 containing callus are really transgenic and that there is no residual agrobacterium in it? The authors write that the “specific protocol was based on our previous research results” (p.5, l.149: ), however, we were unable to find this document (He, S.L.; Wang, Z.; He, D.; Shen, P.; Li, Y.H.; Li, X.J. Chemical staining method of transgenic tree peony callus by GUS gene. 472 Zhengzhou, China, 2019, CN201610078469.6. ) to look at this method.

2.    Did the authors confirm the absence of vir gene in the PoGPT1 containing callus  to make sure of the absence of Agrobacterium?

3.    On what day after the transformation was the expression of the transgene in the calli examined? And on what nutrient medium were the calluses cultivated during this time? Perhaps the PoGPT1 expression shown on Fig. 5 is a consequence of the expression of this gene from the residual plasmid? Why is the expression of  PoGPT1not fixed at all in the control calli? This gene must also be there and also be expressed to some extent, because the translocation of glucose-6-phosphate occurs not only during the process of somatic embryogenesis

4.     Was it actually possible to obtain somatic embryos from this transgenic calli? After all, the work is aimed at solving the problem of obtaining somatic embryos in sufficient quantity and sufficient quality, so does such an overexpression of this gene solve the central problem?

Author Response

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Reviewer 3 Report (New Reviewer)

Song and co-workers observed that overexpression of PoGPT1 induces embryogenesis on tree peony calli, which results very interesting for the vegetative reproduction of this recalcitrant species. Accordingly, the increase of the embryogenesis rate is accompanied by an upregulation of embryogenic-related genes, and an alteration of hormone homeostasis in overexpressing transgenic lines. Although GPT1 is expected to act as a sugar transporter, as reported for some other species, no analysis has been performed with this regard, what would have contributed to the functional characterization of GPT1 in tree peony.  No Mather how, the results presented in the MS are comprehensive, clear, and quiet relevant for tree peony propagation. In my opinion, this manuscript is suitable to be published in Horticulturae if several modifications and further clarifications are included.

Several comments that may improve M&M section are the follows.

Line 101. Buds were in vitro cultured. Where they somehow sterilized? If so, pleas indicate the protocol used.

Lines 119-120. “using the modified CTAB method”, Be more explicit, and/or indicate a reference.

Brief description of tree peony calli protocol would help to further reproduce these results, and to understand the green material management.

I would give some recommendations related to results section.

In my opinion, presenting all in silico data in Table 2 is unnecessary, and does not contribute with valuable information for the manuscript, so I would remove this table from the MS. In silico data can remain along the text in section 3.1 of results, as authors did. Moreover, bioinformatic tools used for this analysis should be indicated somewhere in the MS (in results along the text, or in M&M section).

Figure 3 should be modify in order to make the main message more clear. First, I assume authors stained with DAPI to identify nuclei. If this is the case, please indicate it somewhere in the results along the text. However, in my opinion nuclei in image 3B do not seem to be clearly labeled. In any case, I would recommend to remove this panel, since it is not important for a plastic-localized protein. Next, merged image (figure 3A) is too confusing since bright and 3 fluorescence channels are combined together. I would recommend to remove it, and include a green-red merged image instead. Moreover, co-localization of GFP-PoGPTI and chlorophyll signals is not clear in the presented images. A zoomed in image of red and green panels would help to visualize co-localization. Indeed this result would gain robustness with pixel co-localization quantification using specialized software (i.e. Fiji). Finally, GFP signal seems to be not only in plastids, but also in plasmatic membrane, and cytosol, etc, what should be described in the results section, and further discussed.

Lines 258-260. Authors state that overexpression of PoGPT1 results on an crease of embryogenic cell masses, which is seen in figures 6A-C. These images shows that PoGTP1-derived calli present 4 clusters (black arrows on panel C), whereas only 2 were detected on both control samples (black arrows on panels A and B). Assuming that several calli where dissected, and different sections where obtained, I would encourage authors to show quantitative data regarding this finding. These data could accompany the presented images.

Panels a, b, c (non italic) on figure 6 are not described along the text, and they do not contribute to the general findings on this manuscript, therefore I would recommend to remove them from the figure.

Authors show embryos emerging from calli from PoGTP1 in figure 6 panel c. However, no apparent embryos appear on calli neither from CK nor empty pGREEN (panels a, b). This should be discussed, since authors described a low (but not zero) embryogenesis in wild type plants.  If this is not the case, please point with arrowheads (or other symbol) embryos in these control calli.

For figure 7, statistical analysis indicating whether the observed differences are significant should be included. Moreover, Figure 7 panel A is not described along the result section. Finally, on line 282-284. At 5 days, BL content seem to be slightly higher (or equal) in both genotypes. Authors sated these are lower along the time course, except for time 20 days.

 Discussion can be improved. These are my suggestions.

Authors indicate a high prevalence of transmembrane domains in PoGFP1 according to in silico analysis. However, membrane localization does not seem to be the case for GFP-PoGTP1 heterologous expression and confocal analysis. Can authors reference some previous work that supports this fact?

In the discussion section (lines 349-351) authors state that PoGPT1 is related to auxin polar transport. However, not PoGPT1 but AtGPT1 was studied. This should be modify in the text. In any case, and regarding to auxin accumulation, GPT1 has been shown to be related to polar transport of auxin (ref 26), but no with its accumulation. Is there any reported evidence of it? If this is the case, it should be indicated, and the reference included in the MS.

Finally, results presented in this MS do not support the model shown in figure 9. According to this model, overexpression of PoGPT1 independently activates embryogenesis-related genes, and alters hormone homeostasis. However, no analysis has been done to stablish whether PoGPT1 directly alters embryogenesis-related genes, or whether this upregulation is due to the alteration of the hormone homeostasis within the calli. Therefore, a new branch on this diagram including the possibility of this indirect effect should be included.  

Is there any evidence of impairment of sugar accumulation on GPT1 overexpression in any species, as described for gpt1 mutants in Arabidopsis? If so, pleas discuss it aliong the discussion section.

Few comments related to references are:

Lines 148-149. For peony transformation protocol authors refer to ref 34, what seems to be a patent.  However, I have problems reaching this protocol. Please, clarify this fact (describe briefly the protocol in this paper, or include a link for the patent in the reference list).

References for the bioinformatic tools used to obtain in silico data should be included.

Some further clarifications along the text to make it more readable are:

Lines 91 to 94. It is a bit confusing this paragraph for researchers that are not familiar with this species. Pleas clarify this.

Line 213: “subcellular localization vector” is an awkward way to say “GFP-expressing vector to analyze subcellular localization” or similar.

Line 220: FITC, TD and TRITC, please indicate what these words stand for.

Line 251. Please clarify what “positive identification” in Figure 5 A means.  

Authors measured the ratio of IAA and some other hormones in Figure 8, and these are described on lines 289-292.  The way authors describe these results is, however, a bit abrupt, and misleading for readers. I would recommend to briefly explain the importance of this ratios for embryo development, and why authors decided to calculate them.

 Author Response

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Round 2

Reviewer 1 Report (New Reviewer)

1) Figure 4 is still lack of statistic (letters indicate significant differences).

2) Line 99-100  Authors were asked in the first round of review to demonstrate zygotic embryo development. Here they cite publication (33) however this is a Master's thesis and not available in English. Thus, please describe embryo development and attach photos, so as to explain why exactly these days you took for analysis (presented on Fig. 4).

3) Line 149 Authors didn't explain why subcellular localization was made on N. benthamiana. The reference citted here is not available. Please check the citation.

4) Line 190. Please explain how many sections were analyzed. 

5) Line 232-240 I ask the authors to explain what substances could give red autofluorescence in the plasmatic membrane and cytosol in the leaf???

6) Figure 3 In pictures A and B you can't see that GFP-PoGPT1 construct precisely overlapped with the red autofluorecence. Instead, you can see a much stronger signal covering much more areas/cellular structures for GFP-PoGPT1. 

Author Response

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Reviewer 2 Report (New Reviewer)

It remains unclear and undiscussed why the expression of PoGPT1 was not fixed at all in the control calli? This gene
must also be there and also be expressed in these calli to some extent, because the translocation of
glucose-6-phosphate occurs not only during the process of somatic embryogenesis

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Reviewer 3 Report (New Reviewer)

Song and co-authors presented an improved version of the manuscript entitled “Functional analysis of Glucose-6-phosphate translocator PoGPT1 in embryogenic callus formation of tree peony”. It would have been easier for second correction to include the changes made in their “response to referee letter”, or directly in the manuscript highlighting the changes they have introduced in the new version.  Although authors corrected and improved several aspects of the original MS, there are still some issues that should be improved for its final publication in Horticulturae.

 Figure 3 has been improved, and now it is easier to visualize the PoGPTI and plastids co-localization. However, I have some further comments.

-       Newly introduced text for PoGPTI localization should be revised. Both, lines 230-232, and lines 240-242. Message is clear, but English can be improved.

-       Zoomed in image would help to visualize cell localization.

-       Regarding PoGPTI localization, authors indicate it is localized in plastids due to co-localization with chlorophyl autofluorescence signal. They also state that PoGPTI is localized in vacuoles and chloroplast, but no evidence in figure 3 is shown. It would help to indicate vacuole and chloroplast localization with arrow, asterisks, or someway similar.

-       Also, as I indicate previously, quantification of co-localization of PoGPTI and red signal would help to confirm plastid localization.

-       Bright field somehow confuses green- and red-channels merged image. I would recommend removing it, since it does not add valuable information.

-       PoGPTI clearly shows transmembrane domains, but no membrane localization has been described according to figure 3. This fact should be discussed. Otherwise, including a transmembrane domain analysis in figure 2D is somehow misleading in the MS.

 As I state in my previous revision, results shown in this MS does not support the model shown in figure 9. Authors indicate that PoGPTI overexpression independently alters gene expression and hormone homeostasis, but alteration of hormone accumulation could a consequence of gene activity modification, or vice versa.

 Figure 7b and figure 8, authors indicate one of the genotypes is PsGPTI.

 Author Response

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Round 3

Reviewer 3 Report (New Reviewer)

Dear authors, thanks for considering my suggestions. I hope they were useful. Congratulations for your pub.

Best regards

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

The topic under study is interesting and significant for Peony species. The authors have made great efforts to approach the process of somatic embryogenesis. However, their results cannot be published in this form.

I would suggest that the authors be more analytical in presenting and discussing their observations in the future.

 There are some suggestions:

 1.      Explain which species actually was examined. In the abstract and introduction, it is written that it was Tree peony (Paeonia suffruticosa), however, in the description of the material and methods authors said that the material of Paeonia ostii was used. This causes confusion in the interpretation of the results and their discussion later on. It is known that Paeonia genus is characterised by genetic diversity with relatively rich germplasm resources at the morphological, cytological (chromosome), physiological, biochemical and molecular levels. Based on the presented data in the paper Ji et all (2012.) P. ostii and P. suffruticosa are two different species belong to the Moutan DC, section. What is the latest knowledge regarding the systematics of species of this genus? SE in Paeonia are still fewly reported in some cases. In P. suffruticosa ‘Ren Kaku’, ‘Shima-nishiki’, P. rockii and P. ostii ‘Feng Dan’, direct SE was obtained from immature and mature ZEs (He 2006). It was also found that the SE competency was significantly different with explant genotypes in tree peonies. Incidentally, earlier work on somatic embryogenesis of these two species should be discussed in the presented paper.

 2.      In the material and methods, performed protocol for callus transformation is not specified. Was the transformed callus cleared of the Agrobacterium tumefaciens before expression analysis have been done, i.e. was there a stable callus transformation? Please explain and discuss.

 3.      In the material and methods lines 74 and 75 authors wrote “Pods were collected at 5 d, 60 d, 65 d, 75 d, 90 d, 110 d, from the beginning of podding until full maturity, covering a total 110d.” It means that pods were collected, not excised embryos. Consequently, the results at the Figure 4B cannot present Expression levels in different stages of tree peony embryo development. It could represent only expression levels in different stages of tree peony pods (including immature seeds, with embryos and surrounding tissues – endosperm etc.). 

 4.      In the results, section 3.2 Subcellular Localization of PoGPT1, authors claim “confocal laser microscopy analyses showed that the target protein (GFP-PoGPT1) emitted red fluorescence from the plastid of tobacco” (lines 191 and 192). However, GFP is the green fluorescent protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. In this regard, these allegations need to be explained. In addition, the range of excitation wavelengths as well as the emission wavelengths of the observed protein should be reported in the material and methods. Obtained results should be discussed for each picture presented on Figure 3, ensuring that chlorophyll auto fluorescence does not interfere with GFP fluorescence.

 5.      No somatic embryos are observed in Figures 6A and 6B, so it cannot be argued that “ increased PoGPT1 expression 232 could effectively promote embryogenesis in tree peony calli” lines 231 and 232.

 6.      The role of the PoGPT1 gene in the process of embryogenesis has been discussed, without examining the tissue of isolated embryos in any of the results. The role of the PoGPT1 gene is explained indirectly on the basis of studies in calluses and fruits of this species. Therefore, the claim stated in the title “Glucose-6-phosphate translocator PoGPT1 regulates somatic embryogenesis in tree peony…” is not confirmed by the presented results.

 Minor corrections: 

·       *  Line 200 “35::PoGPT1-GFP vector was infected with N. benthamiana mediated by Agrobacterium”, correct to: “N. benthamiana was infected with 35::PoGPT1-GFP vector mediated by Agrobacterium”

·       *  In the text, be careful when using abbreviations for real time PCR (RT-PCR) and for quantitative reverse transcription PCR (RT-qPCR)

·        * Provide a list of abbreviations

·        * Improve the quality of figures 1, 2, 6

 Author Response

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Reviewer 2 Report

The paper titled 'Glucose-6-phosphate translocator PoGPT1 ……… changes in endogenous hormones’ by Song et. al. has focused on establishing the role of PoGPT1 in somatic embryogenesis of tree peony in relation to endogenous hormonal changes. Although the topic of the study is of significant interest, the following suggestions are for the authors to address in order to further improve the manuscript.

1. Authors are suggested to mention the rationale of selecting the PoGPT1 gene for this study relatively in more detail with literature support in the introduction/discussion section.

2. In Abstract, line no. 26-27, modification in the statement required ‘It coud be used……somatic embryo regeneration system for deeply research.’ Check.

3. Line No. 53, Do you mean GPT1? Please Check. 

4. Line 64-65, Cite relevant study where PoSERK1, PoLEC1, and PoAGL15 were established as key genes for somatic embryogenesis in Tree peony. 

5. In section 2.2, line no. 101, specify which tissue has been selected to extract RNA and C-DNA synthesis.

6. Section, 2.2, Line No. 104, Gene Bank accession no. (ON392713) provided, could not be searched in the database. Please check the accuracy of the same. 

7. Section, 2.7, line no. 156 authors are suggested to provide the suitable citations for the method used.

8. Authors are suggested to follow the sequence in naming figures throughout the manuscript Check line no. 166-180. 

9. Figures 2A, B, C, and D are not very clearly visible and need to be replaced with better images. 

10. Section 3.2, Authors are how much convinced that the result obtained for subcellular localization of PoGPT1 in tobacco will be similar to tree peony too. 

11. Why authors have shown the expression level of PoGPT1 in different tissues in figure 4, needs proper justification. 

12. Whether the data shown in figure 4, is for transformed tissue developed from the somatic embryo of PoGPT1 overexpressed callus or from non-transformed tissue should be mentioned for clarity to the readers. 

13. Whether authors have obtained somatic embryos in this study and observed increased somatic embryogenesis from PoGPT1 overexpressed Calli? If so, provide images of somatic embryos developed. 

14. Whether, the authors have, compared expression levels in PoGPT1 overexpressed callus and untransformed callus? 

15. In sections 3.4 and figure 5, Authors are suggested to include expression analysis data of untransformed tissues too as one of the controls along with CK. 

16. Section 3.6 and figure 7, the Authors are suggested to include untransformed tissues too in consideration as one of the controls to compare hormone content before reaching any conclusion. 

17. Authors are suggested to provide a model diagram based on their findings in this study for regulation of Somatic embryogenesis in tree peony.

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Round 2

Reviewer 2 Report

It's encouraging to note that the authors have attempted to edit the manuscript; however, the authors have been asked to include data for untransformed tissue in the study, which has yet not been done. These data are necessary to support this study, hence I do not feel the paper should be accepted if they are missing.

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