Optimization of Tomato Productivity Using Flowering Time Variants

Round 1

Reviewer 1 Report

The article is generally well written, however, it requires improvement of an experimental part. The major concern is related to gene expression profiling which is lacking at least three key genes, which together with SFT are tomato homologs of Arabidopsis florigen, a FLOWERING LOCUS T gene.

Major comments:

Introduction, line 67

Based on the papers published in recent years, it should be rather mentioned here that there are several (six) FLOWERING LOCUS T homologs in the tomato genome, two of them are hypothetical pseudogenes but the remaining four were evidenced to be transcriptionally active. Moreover, these four active genes have hypothetically opposite influence on flowering time. Namely, SFT is considered as a floral inducer, whereas the other three genes  are expected to be floral inhibitors.

Results, line 271

Expression profiling should include all 4 FLOWERING LOCUS T homologs present in the tomato genome and already evidenced to be transcriptionally active. In my opinion it is obligatory, especially taking into consideration that the study is focused on yield increase using delayed flowering mutants. Thus, three of these FT homologs are hypothesized to participate in flowering delay, because overexpression of these genes in Arabidopsis resulted in late flowering phenotypes. In this study, only flowering activator (SFT) was studied. Moreover, some late flowering mutants presented in this manuscript revealed opposite association between SFT expression and phenotype than expected (high SFT expression and late phenology). Indeed, these mutants may have increased expression of one or more other FT homologs, acting as flowering repressors, and this issue could be addressed here by gene expression profiling. After performing this analysis, appropriate paragraph in the Discussion could be introduced, providing interpretation of the results in the context of other recently published research.

Minor comments

Line 49 – Mutant names, especially those related to gene abbreviations, should be explained at the first use.

Line 141 – Table S1 should be referred here instead of Table S3. Moreover, accession numbers from NCBI or other sequence database should be provided for every target sequence (showing 100% homology to designed primers).

Line 165 – It is not clear whether ten fruits for total soluble sugar content were selected from red ones or from the whole set (red and green).

Line 186 – I suggest rewording to something like that: “revealed the plant weight at least 1.5 times higher than …”

Figure 2 – Column graphs are too small, it could be more feasible to present them larger and placed below the main pictures. The same applies to the small picture in the corner.

Table 1 – Categorization of plants was based on phenotypic data and these data (mean values with standard deviation) should be provided for all studied lines in the supplementary file to visualize differences between particular groups as well as to show some justification why some lines were excluded from further experiments.

Line 301 – replace “ideal” with “optimal”.

Author Response

Reviewer 1

Accommodation to the reviewer’s comments

Thank you very much for the times and efforts as well as the valuable comments. We appreciate that. The comments greatly helped us to improve our manuscript with details of results and descriptions. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have highlighted the changes within the manuscript. Notably, all the changes were edited to ensure that thelanguage is clear by professional editors at Editage.

Reviewer 1

The article is generally well written, however, it requires improvement of an experimental part. The major concern is related to gene expression profiling which is lacking at least three key genes, which together with SFT are tomato homologs of Arabidopsis florigen, a FLOWERING LOCUS T gene.

Major comments:

Introduction, line 67

Based on the papers published in recent years, it should be rather mentioned here that there are several (six) FLOWERING LOCUS T homologs in the tomato genome, two of them are hypothetical pseudogenes but the remaining four were evidenced to be transcriptionally active. Moreover, these four active genes have hypothetically opposite influence on flowering time. Namely, SFT is considered as a floral inducer, whereas the other three genes are expected to be floral inhibitors.

- Response: Thank you for the comments. We totally understand your resonable comments. So we added the following sentences and references in Page 4.

“In addition to SP, three SP5Gs repress florial transition in Nicotiana benthamiana as functional antiflorigens and the SP5Gs expressions were mediated by PHYB in tomato [17]. CR-sp5g sp additively enhanced faster axillary shoot flowering than sp single mutants, resulting in a compact determinate growth habit in tomato [18].”

Results, line 271

Expression profiling should include all 4 FLOWERING LOCUS T homologs present in the tomato genome and already evidenced to be transcriptionally active. In my opinion it is obligatory, especially taking into consideration that the study is focused on yield increase using delayed flowering mutants. Thus, three of these FT homologs are hypothesized to participate in flowering delay, because overexpression of these genes in Arabidopsis resulted in late flowering phenotypes. In this study, only flowering activator (SFT) was studied. Moreover, some late flowering mutants presented in this manuscript revealed opposite association between SFT expression and phenotype than expected (high SFT expression and late phenology). Indeed, these mutants may have increased expression of one or more other FT homologs, acting as flowering repressors, and this issue could be addressed here by gene expression profiling. After performing this analysis, appropriate paragraph in the Discussion could be introduced, providing interpretation of the results in the context of other recently published research.

- Response: Thank you for the comments. We totally understand your resonable comments. So we first count expression values of all SFT homologs from leaf RNA-seq profiling data. SFT and SP5G are expressed in the leaf but not others. Second, we did qRT-PCR analysis with SFT and SP5G. we also confirmed very weak or no expression of SP5G2 and SP5G3 by semi-qRT-PCR using leaf total RNA.

As you suggested, Figure 4b and Figure S2 showing SP5Gs expression in the leaf of flowering time variants. We also added the descritions about SP5Gs expression in results (page 13) and discussion (page 19). All new sentences and changes were highlighted.

 

 

Minor comments

1. Line 49 – Mutant names, especially those related to gene abbreviations, should be explained at the first use.

- Response: Yes, crrently full mutant names are in the first use.

2. Line 141 – Table S1 should be referred here instead of Table S3. Moreover, accession numbers from NCBI or other sequence database should be provided for every target sequence (showing 100% homology to designed primers).

- Response: Yes, we fixed and added gene ID in the table S1.

3. Line 165 – It is not clear whether ten fruits for total soluble sugar content were selected from red ones or from the whole set (red and green).

- Response: We used red fruits for measuring Brix. So we clarified the use of red fruits in line 162.

4. Line 186 – I suggest rewording to something like that: “revealed the plant weight at least 1.5 times higher than …”

- Response: I appreciate your comment. The suggestion is in the line 187~188.

5. Figure 2 – Column graphs are too small, it could be more feasible to present them larger and placed below the main pictures. The same applies to the small picture in the corner.

- Response: Graph and images were enlarged in the figure2

6. Table 1 – Categorization of plants was based on phenotypic data and these data (mean values with standard deviation) should be provided for all studied lines in the supplementary file to visualize differences between particular groups as well as to show some justification why some lines were excluded from further experiments.

- Response: Thank you for this comments. We currently provide all the data of three phenotypic characters. Unfortunately, a few mutants were not coniditionally healthy and accidently missing one of season. Because of those reaosn, we did not used for further experiments. I wish this could be acceptable.

7. Line 301 – replace “ideal” with “optimal”.

- Response: repleaced to “optimal”. Thanks.

Author Response File: Author Response.docx

Reviewer 2 Report

Line 83. Indicate what 'cv' means.

Figure 2 includes inset plots (bar plots) which are very small. Please, consider to remove them or to make another figure with those plots.

In Table S2,  consider to include the units in the headers for 'Flowering time'.

In Table S3, consider to include the units for every variable measured.

In Figure 6b the label of the axis "x" is missing. 

Author Response

Reviewer 2

Accommodation to the reviewer’s comments

Thank you very much for the times and efforts as well as the valuable comments. We appreciate that. The comments greatly helped us to improve our manuscript with details of results and descriptions. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have highlighted the changes within the manuscript. Notably, all the changes were edited to ensure that the language is clear by professional editors at Editage.

Reviewer 2

Are the conclusions supported by the results?

Not applicable

- Response: Thank you for the comment. Revised manuscript has “Conclusions” at the last.

1. Line 83. Indicate what 'cv' means.

- Response: That is mistake. ‘cv’ removed.

2. Figure 2 includes inset plots (bar plots) which are very small. Please, consider to remove them or to make another figure with those plots.

- Response: Thank you for the comment. Current Figure 2 have bigger bar plots and images.

3. In Table S2,  consider to include the units in the headers for 'Flowering time'.

- Response: Thank you for the comment. “Number of leaves in PSM” instead of “flowering time” in Table S2.

4. In Table S3, consider to include the units for every variable measured.

- Response: Thank you for the comment. The units of each trait were included in the table S3.

5. In Figure 6b the label of the axis "x" is missing. 

- Response: Thank you for the comment. Added “Flowering time” as label of x axis in figure 6b.

Reviewer 3 Report

The paper discusses the optimization of tomato productivity in fruits and biomass using flowering time genetic variants. RNA isolation, qRT-PCR, plant dimensions and weights, Brix values, and statistical analysis were used to quantify and correlate the fruit and biomass yields in tomato variants. By isolating 25 delayed flowering variants out of 55 Micro-Tom mutants with late flowering (lf), large plant (lp) and floral homeotic (fh) genes, the authors proposed a fine-tunning optimized model for obtaining a flowering time continuum that assures the increasing of fruit and biomass yields in tomato cultures. Another advantage of the study is that the chosen mutants can be used in detrimental environments and climatic conditions, while the flowering time continuum model could be applied on other types of crops.

Author Response

Accommodation to the reviewer’s comments

Thank you very much for the times and efforts as well as the valuable comments. We appreciate that. The comments greatly helped us to improve our manuscript with details of results and descriptions. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have highlighted the changes within the manuscript. Notably, all the changes were edited to ensure that thelanguage is clear by professional editors at Editage.

Reviewer 4 Report

As a longer duration of the vegetative phase is usually positively correlated with yield, this process needs to consider the length of the agronomic season for any give scenario.

In this study the authors describe a panel of three different groups of flowering mutants in micro tomato. These were chosen based on their effects on growth/floral specification/and late flowering phenotype. These groups were further examined to elucidate the putative correlation between plant biomass, total yield and others related traits with flowering time.  By analyzing these different flowering time mutants, the authors seek to identify tradeoffs between flowering time and yield.

I believe the approach used is interesting, although the idea of tradeoffs between flowering time and yield is not novel. The authors seem to be particularly keen in exploring the role of the florigen system in explaining the observed variability in flowering. This approach revealed potentially novel mechanisms involved in the floral transition. Some specific comments

1. My major concern with this manuscript is that it very difficult to read. I recommend revising the text to make the scope clearer. 
2. The authors should provide more details about the mutants used which are here poorly described (for example genetic description). The supplementary table provided should include a reference to the candidate gene where applicable.
3. Table 1 in the result section is not informative. I suggest substituting with a box plot to show within group variance for the flowering trait examined. Other traits could be also included as late flowering might also impact on leaf biomass. 
4. I am concerned with the definition of fh class. Although this class show alterations in flowering time, the authors should also factor in any effect on yield that depends on aberrant flower development. Except for a brief statement (187 - Seven fh mutants produced abnormal floral organs, such as sepal-like petals and multiple branches (Figure 2 a, d and Table 1) I do not find a more careful description.
5. The authors use bar graphs where the genotypes were arranged by flowering time/group to extrapolate trends of correlations across traits.  I believe there are better ways to represent more accurately and clearly patterns of correlations across traits (within and between groups). I am also surprised why the lf class was not retained in the experiment described in Fig 5.
6. I am not too clear about the conclusions that can be drawn from this manuscript. What is the main driver of yield beside flowering time? Are the authors suggesting that this depends on a different regulatory mechanism of flowering which is not SFT florigen dependent? Maybe this point could be better discussed in the final paragraph based on what is known in the literature.

Finally, in several occasions the appropriate references are missing. Some of them are listed below, but I recommend checking carefully for missing references throughout the manuscript.

Lane 49: ft e hd3a should be include complete mutant names not only abbreviations.   

Lane 51-54: “However, optimized transitional variants in Grain number, plant height and heading date 7 (Ghd7), Heading date 1 (Hd1), and Days To Heading 7 (DTH7), which regulate the florigen pathway, expressed relatively few florigen signals, resulting in minor late-flowering phenotypes with high yielding potential in rice” add the reference

Lane 56: “In ecotypes of Arabidopsis thaliana, genetic variations in key genes regulating floral transition have adapted to seasonal cues at different geographic regions using vernalization and  photoperiod-dependent floral promotion” add the reference, clarify the statement

Lane 407: “Molecularly, the flowering time coupled with plant size and floral organ production could not be only attained using flowering signals, such as florigen activation signals, but might also gain the other flowering signals from floral identity genes and phase transitioning genes. ” add the reference

Author Response

Reviewer 4

As a longer duration of the vegetative phase is usually positively correlated with yield, this process needs to consider the length of the agronomic season for any give scenario.

In this study the authors describe a panel of three different groups of flowering mutants in micro tomato. These were chosen based on their effects on growth/floral specification/and late flowering phenotype. These groups were further examined to elucidate the putative correlation between plant biomass, total yield and others related traits with flowering time.  By analyzing these different flowering time mutants, the authors seek to identify tradeoffs between flowering time and yield.

I believe the approach used is interesting, although the idea of tradeoffs between flowering time and yield is not novel. The authors seem to be particularly keen in exploring the role of the florigen system in explaining the observed variability in flowering. This approach revealed potentially novel mechanisms involved in the floral transition. Some specific comments

1. My major concern with this manuscript is that it very difficult to read. I recommend revising the text to make the scope clearer. 

- Response: We totally understand your comment. In results section, many descriptions get simplified and removed because the paragraph were present in discussion section. All the changes are highlighted in both section.

 

1. The authors should provide more details about the mutants used which are here poorly described (for example genetic description). The supplementary table provided should include a reference to the candidate gene where applicable.

- Response: We understand your comment. All the mutants used in here are not obvisously studied any candidate gene. We try to explain that candidate genes can be studied as further studies in Conclusion section. Because we have focus on phenotypic characters of the mutants to screen phenotypic trends for modelling, we provided phenotic data of all the mutants in Table S2. All the mutants were examined genetic consistance with segregation ratio (not provided).

 

1. Table 1 in the result section is not informative. I suggest substituting with a box plot to show within group variance for the flowering trait examined.

- Response: Thank you for this comment. We understand your comment but we want to simply show how we get late flowering mutants from high-weighted phenotype and floral homeotic phenotype. Additionally, floral homeotic phenotypes is difficult to get quantifiction data because this is a qualitative and arbitrary data.

Other traits could be also included as late flowering might also impact on leaf biomass. 

- Response: Yes, indeed. We examined that high-weighted phenotype and floral homeotic phenotype are highly associated with vegetative biomass and reproductive yield in Figure 5.

 

1. I am concerned with the definition of fh class. Although this class show alterations in flowering time, the authors should also factor in any effect on yield that depends on aberrant flower development. Except for a brief statement (187 - Seven fh mutants produced abnormal floral organs, such as sepal-like petals and multiple branches (Figure 2 a, d and Table 1) I do not find a more careful description.

- Response: Thank you for the comments. We described how we collect the floral homeotic phenotype in Material and Methods, current line 123~125. We try to get heterochronical phenotype in floral and inflorescence organ development. fh mutant strongly increased floral organ or flower number didn’t induced fruit yield amounts in our observation. Flowering time variations among fh mutants were directed the optimal time to get best yield. We discussed that this may be caused by optimal flower productions because of minor increment of reproductive production and late flowering minorly, current line 362-379

 

 

1. The authors use bar graphs where the genotypes were arranged by flowering time/group to extrapolate trends of correlations across traits.  I believe there are better ways to represent more accurately and clearly patterns of correlations across traits (within and between groups).

- Response: Thank you for the great comment. Indeed great comment! Actually, we try to do group assy with independent phenotypic character but bar graphs didn’t enough to show trends of yield related traits because of less genetic resoures in each character, specially fh mutants.

 

I am also surprised why the lf class was not retained in the experiment described in Fig 5.

- Response: We opted to see whether the yield optimum was caused by other clues such as floal homeotic variations or plant weight production, as Fig. 5 show. So we didn’t use lf mutants in the yield assay. Arrangement of the mutants (according to flowering time) directed the pattern of biomass and yield among the lp and fh classes to find out.

 

1. I am not too clear about the conclusions that can be drawn from this manuscript. What is the main driver of yield beside flowering time?

- Response: Thank you for the great comment. Current version has conclusion section. We conclude that our late-flowering variants rearranged with a continuum of flowering time indicated scope for genetic mutants' isolation with high biomass and fruit harvest. Unfortunately, we could say main scope of high yield could be addressed by flowering time screening but could not say other factors in here.

 

Are the authors suggesting that this depends on a different regulatory mechanism of flowering which is not SFT florigen dependent? Maybe this point could be better discussed in the final paragraph based on what is known in the literature.

- Response: Yes, we address your comments (actually questions) in the manuscrpt. Maybe floral homeotic mutants delayed flowering time independently from SFT controlling but not studied in tomato. So we suggest our mutant resources can be identified through mapping using novel sequencing tools in conclusion.

Finally, in several occasions the appropriate references are missing. Some of them are listed below, but I recommend checking carefully for missing references throughout the manuscript.

- Response: we add two more references following.

[17]     Cao, K., Yan, F., Xu, D., Ai, K., et al., Phytochrome B1-dependent control of SP5G transcription is the basis of the night break and red to far-red light ratio effects in tomato flowering. BMC Plant Biol. 2018, 18, 1–12.

[24]     Rothan C., Just D., Fernandez L., Atienza I., Ballias P., L.-C.M., Culture of the Tomato Micro-Tom Cultivar in Greenhouse. Plant Signal Transduct. 2016, 1363, 7–10.

1. Lane 49: fte hd3a should be include complete mutant names not only abbreviations. 

 - Response: added full names in current line 47.

2. Lane 51-54: “However, optimized transitional variants in Grain number, plant height and heading date 7 (Ghd7), Heading date1 (Hd1), and Days To Heading 7 (DTH7), which regulate the florigen pathway, expressed relatively few florigen signals, resulting in minor late-flowering phenotypes with high yielding potential in rice” add the reference

- Response: references added in current line 48-51.

 

3. Lane 56: “In ecotypes of Arabidopsis thaliana, genetic variations in key genes regulating floral transition have adapted to seasonal cues at different geographic regions using vernalization and  photoperiod-dependent floral promotion” add the reference, clarify the statement

- Response: references added in current line 54.

4. Lane 407: “Molecularly, the flowering time coupled with plant size and floral organ production could not be only attained using flowering signals, such as florigen activation signals, but might also gain the other flowering signals from floral identity genes and phase transitioning genes. ” add the reference

- Response: references added in current line 393.

Reviewer 5 Report

The manuscript “Optimization of tomato productivity using flowering time variants” by R. Sujeevan and co-workers describes the productive characterization of a series of tomato microtom (MT) mutants selected for delayed flowering. The authors describe a non-linear correlation between flowering time and yield indicating that mutants with mild delay in flowering time result in higher productivity. The paper thus presents an interesting topic, but is not at present arranged with contents and style sufficient for publication. I give a series of general and specific comments for my opinion.

Introduction: L89-91 say MT is an excellent model …. But I believe MT is also a highly mutagenized plant, affected in hormonal pathways, so the authors should discuss on the suitability of MT to draw general opinions on tomato productivity (making also reference to other authors)

M&M give more details on plant cultivation conditions (eg light), nutrients….

L127-128 the mutants described here are actually not homeotic. Homeotic means with changes of organ identity and this does not pertain to these mutants (see Tab S1). I suggest calling them “Inflorescence and floral mutants”, or alternatively show with more details if they are truly homeotic

L136 please specific clearly whether PCR was quantitative or semi-quantitative

L141 primer info is NOT given in Table3. Also define how many technical and biological reps were adopted.

L171 clarify

Results

Table 1: Those reported are not correlations but percentages

Figure 3 and 5 are not readable (I suggest presenting also fruit number in Fig. 5)

All the work deals with correlations, but no Pearson correlation coefficient is presented. I suggest presenting these coefficients for all the traits measured and for SFT expression (Fig4). In addition, for spring autumn comparison, present ANOVA and correlations.

Why late-flowering mutants have been dropped from Fig. 5?

The model presented in Fig 6b has not been described in M&M

As a discussion section is present, all comments expressed in Results should be removed or minimized

Discussion: L422 the authors mention the concept of resilience, that was not introduced before; please, explain why these mutants should be more resilient

Definitions

Always define abbreviations at the first mention: L71 FD has not been defined, L112 EMS has already been defined, L210 PS already defined

English style: overall the English style should be revised. A number of sentences are not clear due to English form or to their construction (e.g. L22-23, L30-31 “indeterminate traits in floral organs”?, L51, L176 “collective mutants”?, L249 “middle-top dynamics”?, L316-18, L325-326, L337 “indeterminate floral organs”?, L386-388, L405 all the paragraph is difficult to follow

 

 

 

 

Author Response

Reviewer 5

The paper thus presents an interesting topic, but is not at present arranged with contents and style sufficient for publication. I give a series of general and specific comments for my opinion.

 

Introduction: L89-91 say MT is an excellent model …. But I believe MT is also a highly mutagenized plant, affected in hormonal pathways, so the authors should discuss on the suitability of MT to draw general opinions on tomato productivity (making also reference to other authors)

- Response: We understand this comment. Yes, MT has at least three mutations in three major gene which give us the addvantages the followings.

1. Compact plant size and normal fruit setting is suitable for growing in the greenhouse.
2. MT mutant resources are publically available to screen flowering time variants.
3. Because of micro-Tom(compact), it has advantage for screening mutant phenotype and modelling based on phenotyping like we done here.

These advantages were addressed in the introduction (line 88 -96). Reference [23] indicates why MT have been used as model tomato in basic and translational researches.

 

M&M give more details on plant cultivation conditions (eg light), nutrients….

- Response: Thank you for the comment. We minorly added the details about condition of plant growing in line 114-118.

L127-128 the mutants described here are actually not homeotic. Homeotic means with changes of organ identity and this does not pertain to these mutants (see Tab S1). I suggest calling them “Inflorescence and floral mutants”, or alternatively show with more details if they are truly homeotic

- Response: Thank you for the comment. We made a mistake to insert phenotypic information in Table S2. Current Table S2 addressed the phenotypes of fh mutant. We want to keep “floral homeotic” because we try to get heterochronical phenotype in floral and inflorescence organ development.

 

 

L136 please specific clearly whether PCR was quantitative or semi-quantitative

- Response: “Quantitative real-time” instead of “quantitative”

L141 primer info is NOT given in Table3. Also define how many technical and biological reps were adopted.

- Response: Changed to Table 1. Three biological replicates addressed in line 141-142.

 

L171 clarify

- Response: We confirmed all the statistical analysis.

 

Results

 

Table 1: Those reported are not correlations but percentages

- Response: “percentage” instead of “correlations”

Figure 3 and 5 are not readable (I suggest presenting also fruit number in Fig. 5)

- Response: All fonts were enlarged in both figure 3 and 5. Unfortunately, we didn’t collect fruit number. We could expect from total fruits divied by 10 fruit weight but not provided.

 

All the work deals with correlations, but no Pearson correlation coefficient is presented.

- Response: I appreciate this comment which we were missing very critical statistic analysis.

We did bivariate correlation and Non-linear regression analysis using three yield trials for yield modeling. These analysis addressed in Methods (line167-176) and results (line 321-328).

I suggest presenting these coefficients for all the traits measured and for SFT expression (Fig4). In addition, for spring autumn comparison, present ANOVA and correlations.

- Response: Unfortunately, we did not these suggestions that we would like to focus on yield effects according to flowering time. Molecular works could be further studies.

 

Why late-flowering mutants have been dropped from Fig. 5?

- Response: Thank you for this comment. We want to see whether the yield optimum was caused by other clues such as floal homeotic variations or plant weight production, as Fig. 5 show. So we didn’t use lf mutants in the yield assay. Arrangement of the mutants (according to flowering time) directed the pattern of biomass and yield among the lp and fh classes to find out.

The model presented in Fig 6b has not been described in M&M

- Response: Current version has “statistic analysis” in M&M (line167-176).

 

As a discussion section is present, all comments expressed in Results should be removed or minimized

- Response: Thank you for the great comments. We totally understand your comment. In results section, many descriptions get simplified and removed because the paragraph were present in discussion section. All the changes are highlighted in both section.

Discussion: L422 the authors mention the concept of resilience, that was not introduced before; please, explain why these mutants should be more resilient

- Response: I agree with you the sentence could not be addressed by the main text. So deleted “Our new mutants can be utilized for breeding crops that are resilient to land availability, time restrictions, and climatic conditions.”

Definitions

 

Always define abbreviations at the first mention: L71 FD has not been defined, L112 EMS has already been defined, L210 PS already defined

- Response: All comments were accepted and highligted.

English style: overall the English style should be revised. A number of sentences are not clear due to English form or to their construction (e.g. L22-23, L30-31 “indeterminate traits in floral organs”?, L51, L176 “collective mutants”?, L249 “middle-top dynamics”?, L316-18, L325-326, L337 “indeterminate floral organs”?, L386-388, L405 all the paragraph is difficult to follow

- Response: All the changes and English style were edited to ensure that the language is clear by professional editors at Editage.

Especially, following pecific words were changed

“indeterminate development in floral organs” instead of “indeterminate traits in floral organs”

“middle-top curve” instead of “middle-top dynamics”

“floral organs indeterminately” instead of “indeterminate floral organs”

Round 2

Reviewer 1 Report

Comments were more or less accurately addressed. I would like to pay attention that the selection of genes for semi-quantitative PCR based on the presence of their transcripts in RNA-seq from just one time point may be misleading. Genes from flowering regulation pathways are under high transcriptional control and their expression may vary from undetectable values to very high flow in the same plant in different time points.

Moreover, the sentence “Notably, SP5G2 and SP5G3 transcripts were weakly or not detected in all variants' mature leaves (Figure S2)”, present at lines 245-246, seems to be misinterpretation as lines lf8 and lf5 showed at this figure SP5G2 and SP5G3 gene expression levels similar to those detected in wild type. Moreover, some products of SP5G3 at the Figure S2 have different gel migration rates which can indicate the presence of insertion/deletion polymorphism.

Furthermore, please check if Canon camera mentioned at line 125 should not be described as Canon EOS 80D (instead of Canon EDS 80 mentioned there).

Author Response

Reviewer 1

Accommodation to the reviewer’s comments

Thank you for the times and efforts as well as the valuable comments. We appreciate that. The comments greatly helped us to improve our manuscript with details of results and descriptions. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have highlighted the changes within the manuscript.

Comments were more or less accurately addressed. I would like to pay attention that the selection of genes for semi-quantitative PCR based on the presence of their transcripts in RNA-seq from just one time point may be misleading. Genes from flowering regulation pathways are under high transcriptional control and their expression may vary from undetectable values to very high flow in the same plant in different time points.

- Response: Thank you for the comments. We totally agree with your comment. The expression of SFT and SP5Gs show diurnal rhythms. So our comparison may not be sufficient to see the exact variation in each genotype. However, We are not focusing on each genotype but we want to see overall trends of the variations in expression of florigen and antiflorigen. That's why we used one time point in a day to compare SP5Gs expression among the flowering time variants.

1. Moreover, the sentence “Notably, SP5G2 and SP5G3 transcripts were weakly or not detected in all variants' mature leaves (Figure S2)”, present at lines 245-246, seems to be misinterpretation as lines lf8 and lf5 showed at this figure SP5G2 and SP5G3 gene expression levels similar to those detected in wild type. Moreover, some products of SP5G3 at the Figure S2 have different gel migration rates which can indicate the presence of insertion/deletion polymorphism.

- Response: Thank you for this comment. We repeat semi qRT-PCR with two biological replicates. Then, Figure S2 were replaced. The sentence “Notably…” also replaced to the new sentence “Notably, SP5G2 and SP5G3 transcripts were weakly detected in all variants' mature leaves except SP5G2 expression of fh13 and SP5G3 expression of lp10 (Figure S2).”

2. Furthermore, please check if Canon camera mentioned at line 125 should not be described as Canon EOS 80D (instead of Canon EDS 80 mentioned there).

- Response: replaced to “Canon EOS 80D” at line 135. Thanks.

Author Response File: Author Response.docx

Reviewer 4 Report

I am satisfied with the overall corrections made. I still think that the presentation of the results could be made clearer and less redundant. Please consider extensive English editing.

Author Response

Reviewer 4

Accommodation to the reviewer’s comments

Thank you for the times and efforts as well as the valuable comments. We appreciate that. The comments greatly helped us to improve our manuscript with details of results and descriptions. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have highlighted the changes within the manuscript.

 

I am satisfied with the overall corrections made. I still think that the presentation of the results could be made clearer and less redundant. Please consider extensive English editing.

- Response: Thank you for this comment. We try to remove the redundant sentences in results section such as “indicating … late-flowering time” at line 213-214 and “Interestingly, fh8 … PSM” at line 232-2234, and so on. The language is cleared by professional editors at Editage. The language is cleared by professional editors at Editage.

Reviewer 5 Report

The present manuscript is the revised version of a previous submission that was asked for major revisions. Here, most points raised by myself have been addressed and substantially improved. A number of minor points remain to my opinion to be considered, that in part represent objections already raised that have not properly been considered.

It is still not clear to me what a “middle-top-curve” is meant for (L24,L222,L239, L356). It is clear that maximum yield is found in “slightly late flowering” mutants (what slightly means should also be defined), but the relationship with the so called middle-top-curve is not clear.

Still not clear is also “indeterminate development of floral organs” (L29-30, l291). Indeterminate development means that floral organs develop continuously or revert to other organs (eg vegetative?). is that the case?

I still believe the mutants defined homeotic are not truly homeotic. If their main classification is Less number of flowers, Moderate Inflorescence Branching, Heavy inflorescence Branching, Long inflorescence, Large flowers, Less flowers in inflorescence, these to my opinion are NOT homeotic variations. By definition, “mutations in homeotic genes cause displaced body parts”, thus I think the use of homeotic is misleading in this contex. The occurrence of sepal-like petals is indeed a homeotic phenotype but it appeared of secondary importance and not alwasy present in fh mutants

Still the term “collective mutants” is used in L167, but I do not understand its meaning there

The authors state that their expression analysis is quantitative, but still a semi-quantitative approach is shown in Fig S2. Semi-quantitative PCR should be explained in MM.

My suggestion to correlate SFT expression with phenotypic variables has not been considered, but I think it is important to temptatively describe the relationship of this transcript with those variables.

Author Response

Reviewer 5

Accommodation to the reviewer’s comments

Thank you for the times and efforts as well as the valuable comments. We appreciate that. The comments greatly helped us to improve our manuscript with details of results and descriptions. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have highlighted the changes within the manuscript.

The present manuscript is the revised version of a previous submission that was asked for major revisions. Here, most points raised by myself have been addressed and substantially improved. A number of minor points remain to my opinion to be considered, that in part represent objections already raised that have not properly been considered.

1. It is still not clear to me what a “middle-top-curve” is meant for (L24,L222,L239, L356). It is clear that maximum yield is found in “slightly late flowering” mutants (what slightly means should also be defined), but the relationship with the so called middle-top-curve is not clear.

- Response: Thank you for the comments. We accepted all your comments.

In line 24: “an optimal curve” instead of “middle-top-curve”

In line 24: “a certain” instead of “slightly”

In line 222(current L237): “ a bell-shaped curve” instead of “middle-top-curve”

In line 239(current L255): “the optimal curve” instead of “middle-top-curve”

In line 356(current L376): “the bell-shaped curve” instead of “middle-top-curve”

2. Still not clear is also “indeterminate development of floral organs” (L29-30, l291). Indeterminate development means that floral organs develop continuously or revert to other organs (eg vegetative?). is that the case?

- Response: Yes, “indeterminate development” indicates additional productions of floral organs in floral meristems and lateral shoots in axillary meristems. To be clear meaning, “with an additional production of floral organs and lateral shoot” was replaced, instead of “indeterminate development of floral organ and lateral shoots”.

3. I still believe the mutants defined homeotic are not truly homeotic. If their main classification is Less number of flowers, Moderate Inflorescence Branching, Heavy inflorescence Branching, Long inflorescence, Large flowers, Less flowers in inflorescence, these to my opinion are NOT homeotic variations. By definition, “mutations in homeotic genes cause displaced body parts”, thus I think the use of homeotic is misleading in this contex. The occurrence of sepal-like petals is indeed a homeotic phenotype but it appeared of secondary importance and not alwasy present in fh mutants

- Response: Thank you for this comment again. We agree with your comments. However, there are two reasons why we want to use “floral homeotic”.

First, the orginal classification of “floral homeotic” were done by TOMATOMA mutant database. Floral homeotic mutant means the phenotypic mutation in anatomic structure in floral development. We think TOMATOMA phenotyping was included inflorescence structure as floral development. Actually, I also partially agree with TOMATOMA.

 

Second, anantha and fasiflora, two typical floral homeotic mutant, produced extremely branched inflorescences in tomato. After transition to reproductive stage, floral meristem develop not only floral organ but also control inflorescence development. We strongly suggest floral organ development connected with inflorescence aratomy.

 

4. Still the term “collective mutants” is used in L167, but I do not understand its meaning there

- Response: Removed “collective mutants” in L167

5. The authors state that their expression analysis is quantitative, but still a semi-quantitative approach is shown in Fig S2. Semi-quantitative PCR should be explained in MM.

- Response: We added the methods in MM.

6. My suggestion to correlate SFT expression with phenotypic variables has not been considered, but I think it is important to temptatively describe the relationship of this transcript with those variables.

- Response: Thank you for this comment. We agree that correlation SFT expression with phenotypic variables could be important. Unfortunately, the correlations between gene expression(SFT, SF5G) and flowering time were not connected each other as you can see the following data. So we wish not providing the correlation study.

		Relative exp. of SFT	Relative exp. of SP5G
Flowering time	Pearson correlation coefficient	-0.007	0.169
	Significance probability (two-tailed)	0.979	0.517
	N	17	17

 

 

Author Response File: Author Response.docx