Fruit Architecture in Polyamine-Rich Tomato Germplasm Is Determined via a Medley of Cell Cycle, Cell Expansion, and Fruit Shape Genes
Round 1
Reviewer 1 Report
The report by Anwar et al provides evidence for a link between PS homeostasis and expression patterns of fruit shape, cell division and cell expansion genes during early fruit development, suggesting a potential role of PAs in tomato fruit architecture. This was achieved using transgenic tomato plants overexpressing a yeast SPDS. The study is generally well-designed and executed, and data of interest are presented. However, certain points need to be addressed for the ms to reach a level fit for publication (in random order):
Authors used two SPDS lines under the control of CaMV35S, but only one line with a fruit-specific promoter (E8). More than one lines are needed to make sure the effects are not due to transgene positional effects, and going through the original publication (Plant J 2010), it is clear that authors characterized two lines (E8-8 and E8-9 in addition to C4 and C15). Why didn’t the authors also include E8-9 here? How many SPDS isoforms are there in tomato? Figure 3 shows the expression of one, if there are more, then the expression of these should also be examined. I can see that only one housekeeping gene was used for normalization purposes, which is against MIQE standards. Please provide a suitable reference to support the use of ACTIN alone, showing that it is a reliable housekeeping gene with steady expression levels. Format reference list as it is not uniform. General references supporting the function of PAs in plants in the Introduction section [21-30] are in most cases quite dated and should be updated.Author Response
More than one lines are needed to make sure the effects are not due to transgene positional effects, and going through the original publication (Plant J 2010), it is clear that authors characterized two lines (E8-8 and E8-9 in addition to C4 and C15). Why didn’t the authors also include E8-9 here?
We agree with the reviewer that inclusion of E8-9 line would have further added to the observed phenotypes. However, the concern of ‘transgene positional effects’ was overcome by using transgenic lines expressing ySpdSyn under two different promoters. Our finding of similar phenotypes for three independent lines expressing ySpdSyn under the CaMV (2 independent lines) and another under the E8 promoter allow us to interpret our results indicating that obovoid fruit phenotype was not a result of the position effects of transgene insertion. We have used E8-9 in other investigations and it does have obovoid phenotype.
How many SPDS isoforms are there in tomato? Figure 3 shows the expression of one, if there are more, then the expression of these should also be examined.
According to “Sol Genomics Network database”, tomato contains at least 6 spermidine synthase genes. Out of these 6 genes, only one gene (Solyc05g005710) is specific to spermidine synthase activity whereas other five genes (Solyc09g075900; Solyc08g014310; Solyc06g053520; Solyc04g026030) are also considered to have spermine synthase activities. So, to avoid any confusion, transcript levels of only one gene (Solyc05g005710) specific to spermidine synthase activity was quantified.
I can see that only one housekeeping gene was used for normalization purposes, which is against MIQE standards. Please provide a suitable reference to support the use of ACTIN alone, showing that it is a reliable housekeeping gene with steady expression levels.
Since we have previously used actin as the housekeeping gene for determining the relative gene expression (Nambeesan et al., The Plant Journal (2010. 63:836); and Plant Physiology (2012. 158:1034), this was continued to be used in the laboratory. The use of other housekeeping gene(s) is a good suggestion but beyond the scope of the present invrestigation.
Format reference list as it is not uniform.
All references formatted using MDPI referencing style in Endnote software.
General references supporting the function of PAs in plants in the Introduction section [21-30] are in most cases quite dated and should be updated.
Most of the outdated references, except most needed, were either removed or updated with recent citations.
Reviewer 2 Report
The manuscript by Anwar et describes the relationship between polyamine levels and various fruit shape, cell division, and cell expansion genes in regulating tomato fruit architecture. The authors found differential expression of various genes in response to the ySpdSyn transgene expression. Notably, the cellular levels of the bound and free form of PAs correlated with the expression level of these genes. Overall, this study provides evidence for the regulatory role of polyamines as fruit shape modifiers in tomato.
In general, experiments are well designed and carefully performed. The data appeared to be reliable and well-supported conclusions.
Specific points:
1) Figure 1a, what does MG, BR, and Ri denote? It should be described in the legend.
2) Figure 1b, did authors perform significance test for WT vs C4/C15/E88? Also, what do error bars denote? What is n =?
3) Why are error bars missing in fig 2g?
4) Line 143, the sentence” Transgene under the CaMV35 promoter increased SlSpdSyn transcripts at 5 DBA and that under SlE8 at 10 DAP stages of fruit development. Similar results were obtained in C15 fruits” is unclear to me. It appears that in tomato line C4, SlSpdSyn transcript levels are significantly increased at 5 DBP but for C15, it’s 10 DAP. Please clarify.
Minor points:
1) Line 144, DBP and not DBA
2) Line 209, Fig S2 and not S1
3) Line 215, Table S1 and not Figure S1
4) Line 224, DAP and not DAB
5) Line 298, change 2.6 heading to “Transgene Increased Expression of PA Biosynthesis and Catabolic Pathway genes.”
6) Table S2, various developmental “stages”
7) Figure S4, correct “10 DAP”
General comment:
There are a few instances of improper grammar and sentence structure, such as in lines 267, 274, 279, 334, etc.
Author Response
1) Figure 1a, what does MG, BR, and Ri denote? It should be described in the legend.
MG – mature green stage; BR – breaker stage; Ri – red ripe stage of tomato fruit development. These are now described in the figure legend.
2) Figure 1b, did authors perform significance test for WT vs C4/C15/E88? Also, what do error bars denote? What is n =?
Yes, both ANOVA and Tukey’s Test showed statistically significant variation in fruit shape from transgenic lines as compared with WT. Error bars denote standard error of means. ‘n’ represents number of replications whereas each replication contains at least 50 tomato fruits (now also added in figure description).
3) Why are error bars missing in fig 2g?
Cell size ratio was calculating by dividing average size of cells at 20 days after pollination (DAP) (Fig. 2f) to average size of cell at 10 DAP (Fig. 2e). So, standard error could not be calculated as the cell size ratio was based on mean values.
4) Line 143, the sentence” Transgene under the CaMV35 promoter increased SlSpdSyn transcripts at 5 DBA and that under SlE8 at 10 DAP stages of fruit development. Similar results were obtained in C15 fruits” is unclear to me. It appears that in tomato line C4, SlSpdSyn transcript levels are significantly increased at 5 DBP but for C15, it’s 10 DAP. Please clarify.
Yes, this is right. Correction was incorporated in the manuscript. Thanks.
Minor points:
1) Line 144, DBP and not DBA
Corrected.
2) Line 209, Fig S2 and not S1
Corrected.
3) Line 215, Table S1 and not Figure S1
Corrected.
4) Line 224, DAP and not DAB
Corrected.
5) Line 298, change 2.6 heading to “Transgene Increased Expression of PA Biosynthesis and Catabolic Pathway genes.”
Changed.
6) Table S2, various developmental “stages”
Corrected.
7) Figure S4, correct “10 DAP”
Corrected.
General comment:
There are a few instances of improper grammar and sentence structure, such as in lines 267, 274, 279, 334, etc.
Corrected.
Round 2
Reviewer 1 Report
Ok with the way authors handled my concerns.
