Transcriptomic Profiling of Pomegranate Provides Insights into Salt Tolerance
, Jinping Wang 1,2, Mengmeng Gu 3,* and Zhaohe Yuan 1,2,*Round 1
Reviewer 1 Report
The paper deals with pomegranate response to salinity stress. The authors want to elucidate the molecular responses at transcriptomic level and they found 2,255 DEGs (differentially expressed genes), some genes up-regulated and others down-regulated. These DEGs are tissue and time specific . Moreover, the expression pattern was different between roots and leaves. A comparison between the two tissues was done and a set of 15 genes were chosen to confirm RNA-seq data. The authors conclude that the study will help future breeding of pomegranate.
The paper deals with pomegranate response to salinity stress. The authors want to elucidate the molecular responses at transcriptomic level and they found 2,255 DEGs (differentially expressed genes), some genes up-regulated and others down-regulated. These DEGs are tissue and time specific . Moreover, the expression pattern was different between roots and leaves. A comparison between the two tissues was done and a set of 15 genes were chosen to confirm RNA-seq data. The authors conclude that the study will help future breeding of pomegranate.
More in details:
The authors need to show the pictures of treated and untreated plants. Did the plants behave the same? Did the plants show any symptom related to the stress? 200mM NaCl is a very high concentration of salt. The experiment on RNA expression after a stress treatment have to be performed on a short (less than 12 hours) time of stress since many of the stress related genes are induced during the first hours. In this paper the first time-point is three days that is too much. Indeed, after this period the system is already “adapted” to the new environment, so most of the up or down regulated genes are missed. There is plenty of literature dealing with this topic. This point is particularly delicate considering that the authors would like to use their data for a future breeding. The early response genes are fundamental for the selection of a tolerant genotype. Moreover to support the RNASeq data I think it is necessary to perform some physiological experiments, such as the measurement of some ion concentrations (Na,K and Ca), proline content, enzyme activities related to the response (catalase, ascorbate peroxidase….), all these compounds are well known to mediate the salt stress tolerance.
For these reasons I think that the study here presented is weak and not sufficiently supported.
Author Response
Response to Reviewer 1 Comments
Point 1: The authors need to show the pictures of treated and untreated plants. Did the plants behave the same? Did the plants show any symptom related to the stress? 200mM NaCl is a very high concentration of salt. The experiment on RNA expression after a stress treatment have to be performed on a short (less than 12 hours) time of stress since many of the stress related genes are induced during the first hours. In this paper, the first time-point is three days that is too much. Indeed, after this period the system is already “adapted” to the new environment, so most of the up or down regulated genes are missed. There is plenty of literature dealing with this topic. This point is particularly delicate considering that the authors would like to use their data for a future breeding. The early Response genes are fundamental for the selection of a tolerant genotype. Moreover to support the RNASeq data I think it is necessary to perform some physiological experiments, such as the measurement of some ion concentrations (Na,K and Ca), proline content, enzyme activities related to the Response (catalase, ascorbate peroxidase….), all these compounds are well known to mediate the salt stress tolerance.
For these reasons, I think that the study here presented is weak and not sufficiently supported.
Response 1: I included a picture of treated and untreated plants in the supplemental material as Figure S1. In the picture, there was not visible difference between the treated and untreated plant. RNA expression pattern of 12 hours stress is a good suggestion, however, the early response (during the first hours) genes were not the focus of the current study. We were more interested in the short-term adaptation process of up- or down-regulated genes from 3 days to 6 days. Pomegranate is considered as a salinity-tolerant plant. Physiological responses to salt stress were determined in our previous study, which showed that the physiological and biochemical parameters (such as chlorophyll content, malondialdehyde content, proline content, soluble protein content and enzyme activities) had no significant changes at medium salinity (0.4%) after 7 days (Liu, C.; Yan, M.; Huang, X.; Yuan, Z. Effects of salt stress on growth and physiological characteristics of pomegranate (Punica granatum L.) cuttings. Pak. J. Bot. 2018, 50, 457-464.).
Reviewer 2 Report
Abstract
Lines 26-27: The sentence: "To confirm....PCR" needs to be rewritten.
Materials and Method
Line 80: Please state the total weight of Perlite and Peat in each pot.
Line 82: Please clarify whether the plants were waterred with fresh water during the growing and experimental periods. How often the plants were watered with fresh water?
Line 85: The author should give more information about the salinity stress experiment: e.g How much (what is the volume of) half-strength Hoagland’s solution supplemented with 200 mM NaCl were irrigated to each pot? Were all roots contacted directly with NaCl in ½ Hoagland’s solution while being fertigated? Were all pots in the experiment watered the same amount of NaCl in 1/2 Hoagland’s solution?
Lines 85-87: The sentence "...,and the roots..." needs to be rewritten to indicate that 45 plants were randomly selected, 15 control, 30 subjected to stress and samples were collected at 2 time points
Line 89: These samples were for RNA extraction. what did authors mean by further experiments?
Line 96-105: Illumina has a standard library preparation protocol, did the authors use this protocol or a custom one? Please clarify. Important steps like adaptor/barcode ligation is missing in this section. Also please review the use of English words "At last" and "Finally" in lines 102 and 104.
Lines 118-121: Was this supposed to be done after cufflinks?
Results
Please provide photos of plants at beginning, 3 days and 6 days stress time points and give information about whether morphological changes have been observed under salt stress at the 2 time points
Line 144: Are these RNAseq libraries or cDNA libraries?
Line 147: we can use in total or totally, not in totally.
Table 1 needs to show that the percentages listed in lines 150 -151 are from clean reads i.e. from 155Gb not 519Gb data. Please add a column of clean reads, discarded to the Table.
All figures in the manuscript are not high quality. Please provide high quality figures. Especially Figure 2 and 4 are not clear and generally unable to read details on y and x axis.
Line 217: please add a reference for STEM
Figure 5 a) is not a result from this study therefore I suggest to remove Figure 5a and make Figure 5c bigger and clearer. The same suggestion for Figure 6a.
For qRT-PCR section please provide a Figure showing relative expression level. The authors can use bar graphs to present the relative expression level of the genes normalizing to the expression of house keeping genes.
Discussion
Lines 327-328: the authors did not provide a tool, the RNAseq was used as a tool in their study. Therefore the sentence needs to be rewritten.
Lines 329-330: The authors mentioned that 5,396 novel genes were identified but no expression profiles of these genes were presented. The authors could have selected a few of these genes for qPCR and associated them with the salinity tolerance for more novel results.
Lines 333-334: The sentence: " the coding and noncoding sequences in reference genome were annotated incorrectly" may be an over-claim. Do the authors have evidence for this claim? I suggest the sentence be rewritten so that it is understood as an prediction rather than a confirmation.
Author Response
Dear reviewer,
All authors would like to thank for your time and effort in improving this manuscript. The following is a point-by-point response to each comments.
Please see the attachment.
Please let me know if you have any questions.
Sincerely,
Cuiyu Liu
Responses to Reviewer 2 Comments
Point 1: Lines 26-27: The sentence: "To confirm....PCR" needs to be rewritten.
Response 1: The sentence has been rewritten as “Fifteen genes were selected to confirm the RNA-seq data using qRT-PCR.”
Point 2: Line 80: Please state the total weight of Perlite and Peat in each pot.
Response 2: The total weight of perlite and peat in each pot is 1.5 ± 0.1 Kg.
Point 3: Line 82: Please clarify whether the plants were watered with fresh water during the growing and experimental periods. How often the plants were watered with fresh water?
Response 3: As stated in L82, the plants were fertigated weekly with ½ Hoagland’s nutrient solution during the growing period, and then they were fertigated once with 200 mM NaCl in ½ Hoagland’s solution during the experiment period (6 days).
Point 4: Line 85: The author should give more information about the salinity stress experiment: e.g How much (what is the volume of) half-strength Hoagland’s solution supplemented with 200 mM NaCl were irrigated to each pot? Were all roots contacted directly with NaCl in ½ Hoagland’s solution while being fertigated? Were all pots in the experiment watered the same amount of NaCl in 1/2 Hoagland’s solution?
Response 4: We fertigated the plants once with 500 ml of ½ Hoagland’s solution containing 200 mM NaCl. A saucer was placed under containers to collect leachate solution during the experiment period. All pots in the experiment watered the same amount of NaCl in 1/2 Hoagland’s solution. All roots were not dipped in solution but directly contacted with saline soil.
Point 5: Lines 85-87: The sentence "...,and the roots..." needs to be rewritten to indicate that 45 plants were randomly selected, 15 control, 30 subjected to stress and samples were collected at 2 time points
Response 5: We have rewritten this paragraph based on the reviewer’s comment: ‘At the beginning of experiment, forty-five plants were randomly selected and divided into 3 groups, fifteen plants per group. The roots and leaves of the first group were harvested as control samples (T0R and T0L, respectively). The other two groups of plants were fertigated once with 500 ml of ½ Hoagland’s solution containing 200 mM NaCl. A saucer was placed under each container to collect leachate solution during the experiment period. The roots and leaves of the second group on day 3 (T1R and T1L) and the third group on day 6 (T2R and T2L) were harvested later. Samples from five plants in the same group were pooled together as a replicate due to the small amount of biomass. All samples were immediately frozen in liquid nitrogen and then stored at -80 °C.’
Point 6: Line 89: These samples were for RNA extraction. what did authors mean by further experiments?
Response 6: Yes, these samples were for RNA extraction.
Point 7: Line 96-105: Illumina has a standard library preparation protocol, did the authors use this protocol or a custom one? Please clarify. Important steps like adaptor/barcode ligation is missing in this section. Also please review the use of English words "At last" and "Finally" in lines 102 and 104.
Response 7: We have rewritten this paragraph and clarified the question, and modified as Line 114-132.
Point 8: Lines 118-121: Was this supposed to be done after cufflinks?
Response 8: Thanks, we used the StringTie instead of cufflinks.
Point 9: Please provide photos of plants at beginning, 3 days and 6 days stress time points and give information about whether morphological changes have been observed under salt stress at the 2 time points
Response 9: I included a picture of treated and untreated plants in the supplementary Figure S1 and from the picture, there was not visible difference between the treated and untreated plant.
Point 10: Line 144: Are these RNAseq libraries or cDNA libraries?
Response 10: Thanks, these libraries are cDNA library.
Point 11: Line 147: we can use in total or totally, not in totally.
Response 11: Revision has been made based on the reviewer’s comment.
Point 12: Table 1 needs to show that the percentages listed in lines 150 -151 are from clean reads i.e. from 155Gb not 519Gb data. Please add a column of clean reads, discarded to the Table.
Response 12: All the percentages listed in this report are from clean reads. Line 133 “All the downstream analyses were based on clean data with high quality.”
Point 13: All figures in the manuscript are not high quality. Please provide high quality figures. Especially Figure 2 and 4 are not clear and generally unable to read details on y and x axis.
Response 13: We have upgraded figures in TIFF format.
Point 14: Line 217: please add a reference for STEM
Response 14: Thanks. We added the reference for STEM in Line 196.
Point 15: Figure 5 a) is not a result from this study therefore I suggest to remove Figure 5a and make Figure 5c bigger and clearer. The same suggestion for Figure 6a.
Response 15: The pathway (5a) was included to facilitate the audience’s understanding of the roles of the genes of interest (PYI, PP2C and SnRK2).
Point 16: (Fig. 7) For qRT-PCR section please provide a Figure showing relative expression level. The authors can use bar graphs to present the relative expression level of the genes normalizing to the expression of house keeping genes.
Response 16: We agree that the bar graph is a good choice, however, in addition to include information included in a bar graph, the scattered data points of as many as 15 genes in this graph could also show the correlation between RNA-seq data with qRT-PCR data, R2 ≥ 0.85 (Figure 7), whichimplies the reproducibility and accuracy of the RNA-Seq results.
Point 17: Lines 327-328: the authors did not provide a tool, the RNAseq was used as a tool in their study. Therefore the sentence needs to be rewritten.
Response 17: ‘In this study, we used the RNA-Seq approach as a powerful tool to elucidate the molecular responses to salt stress in pomegranate.’
Point 18: Lines 329-330: The authors mentioned that 5,396 novel genes were identified but no expression profiles of these genes were presented. The authors could have selected a few of these genes for qPCR and associated them with the salinity tolerance for more novel results.
Response 18: The novel genes were identified from transcripts after mapping to genome. The different expression novel genes were also defined as DEGs and employed to functional analysis, so we didn’t show the expression profiles. The qRT-PCR of these novel genes is a good idea, but the results mainly confirm the veracity of RNA-seq data.
Point 19: Lines 333-334: The sentence: " the coding and noncoding sequences in reference genome were annotated incorrectly" may be an over-claim. Do the authors have evidence for this claim? I suggest the sentence be rewritten so that it is understood as an prediction rather than a confirmation.
Response 19: ‘the coding and noncoding sequences in reference genome may have been annotated incorrectly’.
Author Response File: 
Author Response.pdf
Reviewer 3 Report
The manuscript "Transcriptomic profiling of pomegranate provides 2 insights into salt tolerance" is an interesting area of research on transcriptomics. Clear objectives, methodology and data analysis. The results were presented in details with appropriate interpretation. Relevant discussion and conclusion. Few language edition were observed. The figure captions need to be descriptive. Figure 1 has four parts but not clearly labeled as A, B, C, D.
Figure 2 not readable at all, it has also four parts not described well.
Figure 3 has four parts but not described in the captions
Figure 4 not readable at all. it needs to increase the fonts in the figure.
Figure 7 the same as above.
Adjustment of font size for captions of all the figures need to be improved to the font size of the text.
Author Response
Dear reviewer,
All authors would like to thank for your time and effort in improving this manuscript. The following is a response to each comment.
Responses:The figures were not readable because they were compressed in the WORD file. All figures were also provided separately in TIFF format, which is much more readable. We modified these figures according to the author instruction of Agronomy and added the captions of each part of figure.
Please let me know if you have any questions.
Sincerely,
Cuiyu Liu
Reviewer 4 Report
The paper by Liu et al. presents data of a high scientific soundness. My only comment concerns the quality of picture resolution – this should be improved.
Author Response
Dear reviewer,
All authors would like to thank for your time and effort in improving this manuscript. The following is a response to each comment.
Response:All figures were also provided separately in TIFF format, which is much more readable.
Please let me know if you have any questions.
Sincerely,
Cuiyu Liu
