Expression and Functional Analysis of the PaPIP1-2 Gene during Dormancy and Germination Periods of Kernel-Using Apricot (Prunus armeniaca L.)
Round 1
Reviewer 1 Report (Previous Reviewer 2)
Comments and Suggestions for AuthorsReview Forests 2428763 revised version
I would recommend accepting the revised version of the manuscript ”Expression and functional analysis of the PaPIP1-2 gene during dormancy and germination periods of kernel-using apricot (Prunus armeniaca L.)” for publication since many problems were eliminated in the revised paper. However, some meticulous corrections still need to be made, in particular.
Line 90 “In” is missed
Line 107 “2.2. Structural Features of Putative PaPIPs” should be changed to “2.2. Analysis of Structural Features of Putative PaPIPs” since the passage below does not contain the results
Lint188 “strains” to “strain” as only one strain GS115 is mentioned
Lines365-370: In the caption to Figure 6, it is necessary to mention the PaPIP1-1 and PaPIP 2-3 constructs, the analysis of which confirms the results of yeast studies.
Fig 7b: WT is absent
Lines482-484.”(f) Expression levels of cold resistance genes including AtPUB26. (g) Expression levels of cold resistance genes including AtBTF3L. (h) Expression levels of cold resistance genes including AtEBF1-1-1 (i) Expression levels of cold resistance genes including AtRAV1” should be changed to “Expression levels of cold resistance genes including (f) AtPUB26, (g) AtBTF3L, (h) AtEBF1 and (i) AtRAV1”
Line 528 please, delete “illustrates the”
Line 537 please, delete “displays a”
Comments on the Quality of English LanguageMinor editing of English language required
Author Response
Reply to the reviewer
Dear Editor of Forests,
Our original manuscript, titled “ Expression and functional analysis of the PaPIP1-2 gene during dormancy and germination periods of kernel-using apricot (Prunus armeniaca L.)” (Manuscript ID: 2428763), has been assessed by reviewers of Forests.
The paper was revised according to your and other reviewer’s suggestions. We hope that our revised manuscript now meets the requirements for publication in Forests. Here, we resubmit our revised manuscript for publication in Forests. The revisions are listed below in detail. Important modifications are marked in green in revised manuscript.
Review Forests 2428763 revised version
I would recommend accepting the revised version of the manuscript ”Expression and functional analysis of the PaPIP1-2 gene during dormancy and germination periods of kernel-using apricot (Prunus armeniaca L.)” for publication since many problems were eliminated in the revised paper. However, some meticulous corrections still need to be made, in particular.
- Line 90 “In” is missed
Response:
In Line 94, we wrote, “In this study,“
- Line 107 “2.2. Structural Features of Putative PaPIPs” should be changed to “2.2. Analysis of Structural Features of Putative PaPIPs” since the passage below does not contain the results
Response:
In Line 111, we wrote, “2.2. Analysis of Structural Features of Putative PaPIPs“
- Line188 “strains” to “strain” as only one strain GS115 is mentioned
Response:
In Line 195, we wrote, “GS115 yeast strain“
- Lines365-370: In the caption to Figure 6, it is necessary to mention the PaPIP1-1 and PaPIP 2-3 constructs, the analysis of which confirms the results of yeast studies.
Response:
In Line 364-365, we wrote, “the pGAPZA::PaPIP1-2 (Figure S4), the pGAPZA::PaPIP1-1, or the pGAPZA::PaPIP2-3 recombinant expression vectors”.
In Line 565-567, we wrote, “Figure S4. vector map of pGAPZA. EcoR Iå’ŒXho I. According to the pGAPZA vector map and the sequence of the PaPIP1-2 gene, EcoR I and XhoI were designed as insertion sites.”
the analysis of which confirms the results of yeast studies?
Response:
We think the analysis of cold resistance of PaPIP1-2 transgenic Arabidopsis plants (Figure 7) confirms the cold resistance results of yeast studies.
- Fig 7b: WT is absent
Response:
In Fig 7b, expression levels of PaPIP1-2 quantified using qRT-PCR in two distinct PaPIP1-2 transgenic lines (PIP1-2 OE-1 and PIP1-2 OE-2). We think that PaPIP1-2 gene was isolated from Prunus armeniaca L, thus we could not detect the expression of apricot gene in wild type Arabidopsis.
- Lines482-484.”(f) Expression levels of cold resistance genes including AtPUB26. (g) Expression levels of cold resistance genes including AtBTF3L. (h) Expression levels of cold resistance genes including AtEBF1-1-1 (i) Expression levels of cold resistance genes including AtRAV1” should be changed to “Expression levels of cold resistance genes including (f) AtPUB26, (g) AtBTF3L, (h) AtEBF1 and (i) AtRAV1”
Response:
Thanks to reviewer for this guidance. In Line 502-503, we wrote, “Expression levels of cold resistance genes including (f) AtPUB26, (g) AtBTF3L, (h) AtEBF1 and (i) AtRAV1.”
- Line 528 please, delete “illustrates the”
Response:
In Line 548, we delete “illustrates the”.
- Line 537 please, delete “displays a”
Response:
In Line 557, we delete “displays a”.
Reviewer 2 Report (Previous Reviewer 3)
Comments and Suggestions for AuthorsThe author has responded appropriately to the reviewers' comments.
Author Response
Thank you for your review
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe objectives of the study are not clear, all the experimental results are not strongly relevant and the authors did not characterize functionally the protein.
Abstract:
Line 16: “Here, the function…” - You do not characterize the function of the gene.
Line 17: “Our results show that PIP2 protein expression” – PIP2?? Expression is for genes, not proteins.
Line 19: “low temperature strain” - You mean stress? And is chilling or freezing?
Line 20: “The mRNA profile of every PaAQP that showed coexpression with genes related to invulnerability against low temperature strain remained according to all stages of P. armeniaca flower bud development.” – Is not usual to say mRNA profile, you can say mRNA abundance or gene expression profile. Is not invulnerability to a stress, is resistance or susceptibility. And this sentence is too confusing, rewrite.
Line 26: “protein enrichment in yeast” – This is not a used expression.
Line 27: You need to write the extensive name of MDA in your first mention.
Introduction:
The English is agonizing.
Line 36: “that participate in water molecule” - ?? Participate or allow? Please be careful with the wording.
The objectives have a flaw as the authors do not mention any valid reason to study specifically the PIP1.2.
Line 89: This last sentence is not adequate for the introduction as it is a conclusion.
MM:
All this section is badly written and confusing.
Line 206: “Following a whole day at -20°C and shaking (200 rpm, 30°C)???? Is -20 ºC or 30 ºC??
Line 234: “evidenced the top expression outcomes throughout various periods of ED, SP, and GS (Figure S1)” – albeit PaPIP1.2 showed a high expression in the sampled material, they are no top. They are in line with other aquaporins of the cluster 1.
Why do you have this here and not in the objectives if is a sequential work??
There is no figures2
There are no supplemental tables!!
Line 309: “…and can accelerate the development of freezing tolerance [27].” - What this even mean?
Line 307 to 314: This is for the discussion section not result section.
Line 328: “regulation of experience-dependent behavioral modifications in mice [30].” – Is this relevant?
A lot of formatting errors.
Line 343: “Notably, all six gene pairs showed highly consistent expression patterns with PaPIP1-2 across all four evaluated phases.” – notably the expression pattern of these genes is not so highly consistent with PaPIP1.2 expression. Other exercise that is important is to analyze if the expression of those genes is high or low because if you have a similar expression pattern but with near 0 expression the gene will not have importance.
I cannot understand the relevance of performing a -20 ºC freeze tolerance assay in yeast. Why did you studied the other PIPs?
Line 360: “…after exposure to -20°C had a noticeably enhanced growth compared to cells that had undergone…” – I cannot agree with this sentence. In the solid medium is not “noticeably enhanced”.
Again, in the Arabidopsis tolerance assays the authors say that the results are evident when they are not. And these critical results are not quantified, they are an “evidence” that the plants “showed”. No quantification of nothing.
Comments on the Quality of English LanguageThe english is agonizingly badly written.
Author Response
Reply to the reviewer
Dear Editor of Forests,
Our original manuscript, titled “ Expression and functional analysis of the PaPIP1-2 gene during dormancy and germination periods of kernel-using apricot (Prunus armeniaca L.)” (Manuscript ID: 2428763), has been assessed by reviewers of Forests. The paper was revised according to your and the three reviewer’s suggestions. Extensive editing of the English language was performed by a native English speaker. We hope that our revised manuscript now meets the requirements for
publication in Forests.
Here, we resubmit our revised manuscript for publication in Forests. The revisions are listed below in detail. Important modifications are marked in red in revised manuscript.
Response to reviewer 1
Question: Line 16: “Here, the function…” - You do not characterize the function of the gene.
Response:
Several experiments such as overexpression of PaPIP1-2 in Arabidopsis and yeast strains, gene expression analysis demonstrated that the P. armeniaca PaPIP1-2 gene improves cold stress tolerance and plays a key role in the dormancy and germination stages.
Question: Line 17: “Our results show that PIP2 protein expression” – PIP2?? Expression is for genes, not proteins.
Response:
We apologize for this oversight. On lines 21-22 of revised MS, we have written the following:
“Our findings revealed that the expression of the PaPIP1-2 gene is coordinated during both dormancy and sprouting periods.”
Question: Line 19: “low temperature strain” - You mean stress? And is chilling or freezing?
Response:
We apologize for this oversight. On lines 23-24 of revised MS, we have written the following:
“Through a PaPIP1-2 gene network analysis, we found that it exhibited coexpression with six genes related to low-temperature stress.”
Question: Line 20: “The mRNA profile of every PaAQP that showed coexpression with genes related to invulnerability against low temperature strain remained according to all stages of P. armeniaca flower bud development.” – Is not usual to say mRNA profile, you can say mRNA abundance or gene expression profile. Is not invulnerability to a stress, is resistance or susceptibility. And this sentence is too confusing, rewrite.
Response:
We thank the reviewer for this suggestion. On lines 24-26 of revised MS, we have written the following:
“The mRNA abundance of each PaAQP, which displayed coexpression with genes associated with resistance against low-temperature stress, remained consistent throughout all stages of P. armeniaca flower bud development.”
Question: Line 26: “protein enrichment in yeast” – This is not a used expression.
Response:
We thank the reviewer for this question. On lines 31-32 of revised MS, we have written the following:
“Yeast strains overexpressing PaPIP1-2 demonstrated increased cold resistance, with enriched protein content in the yeast cells.”
Question: Line 27: You need to write the extensive name of MDA in your first mention.
Response:
We thank the reviewer for this question. Besides the abstract, the first mention is on lines 401-404 of revised MS, we have written the following:
“The cold resistance physiological characteristics of the PaPIP1-2-transformed plants were further analyzed by assessing the MDA (malondialdehyde) and proline contents following cold stress.
the first mention in abstract, we also added as follow:
“This improvement was accompanied by a reduction in MDA (malondialdehyde) levels, increased proline accumulation and superoxide dismutase (SOD) activities, and a decrease in ion leakage content compared to that of the WT (wild-type) plants.”
Question: Line 36: “that participate in water molecule” - ?? Participate or allow? Please be careful with the wording.
Response:
We thank the reviewer for this question. On lines 45-47 of revised MS, we have written the following:
“Aquaporins (AQPs) are essential water channel proteins that facilitate the flow of water molecules across cell membranes and aid the passage of other small solutes like H2O2, silicic acid, boric acid, and glycerol [1,2].”
Question: The objectives have a flaw as the authors do not mention any valid reason to study specifically the PIP1.2.
Response:
We thank the reviewer for this question. There are several reason justify the need to study the function of PIP1.2. Firstly, Previous research has demonstrated the important roles of PIP subfamily members in cold acclimation and freezing tolerance. There are some connections between plant cold resistance and dormancy and germination (Li et al., 2020). PaPIP1-2 showed the differential expression in the dormancy and sprouting periods of P. armeniaca, this indicated that PaPIP1-2 may involved and play an important role in dormancy and germination periods of flower buds (Figure 1, Figure S1). Secondly, some typical protein domains exist in the PaPIP1-2 protein, such as MIP domain, Crp-type HTH domain, etc, this showed that PaPIP1-2 protein is potentially involved in crucial biological processes including signal transduction, transcriptional activation, and material transport, and need further research (Figure 2). Lastly, we found that several important proteins such as phosphatase 2C, Leucine-rich receptor-like protein kinase, which involved in cold resistance or germination speed, were potential interacting protein of PaPIP1-2 as confirmed by constructing a protein-protein interaction network. We are curious about the role of PaPIP1-2 gene in plant dormancy and germination, and resistance to cold stress. So other experiments such as transgenic and physiological analysis were taken to prove the function of this gene.
Related References
Li, S.; Wang, L.; Zhang, Y.; Zhu, G.; Zhu, X.; Xia, Y.; Li, J.; Gao, X.; Wang, S.; Zhang, J.; et al. Genome-Wide identification and function of aquaporin genes during dormancy and sprouting periods of Kernel-Using Apricot (Prunus armeniaca L.). Front Plant Sci 2021, 12, 690040.
Question: Line 89: This last sentence is not adequate for the introduction as it is a conclusion.
Response:
We thank the reviewer for this question. On lines 106-108 of revised MS, we have written the following:
“Our findings suggest that the PaPIP1-2 gene potentially plays a crucial role in responding to flower bud dormancy and germinaton, and conferring resistance to various forms of stress such as low-temperature stress.”
Question: Line 206: “Following a whole day at -20°C and shaking (200 rpm, 30°C)???? Is -20 ºC or 30 ºC??
Response:
We thank the reviewer for this question. On lines 216-222 of revised MS, we have written the following:
“In brief, yeast strains containing either the empty vector (pGAPZA vector) or the PaPIP1-2 gene (considered positive) were cultured with shaking (200 rpm) at 30°C for one full day to activate the expression of the PaPIP1-2 protein. Subsequently, a portion of the liquid yeast culture (1 mL) was taken, containing either the pGAPZA::PaPIP1-2 recombinant vector or the pGAPZA empty vector, and subjected to low-temperature treatment (-20°C) for 24 hours at OD600 = 1.0.”
I would like to provide an explanation, yeast strains were cultured with shaking (200 rpm) at 30°C for one full day, This function is to activate the expression of the PaPIP1-2 protein. Then, 1 mL liquid yeast culture was taken and subjected to low-temperature treatment (-20°C), so low-temperature stress was performed.
Question: Line 234: “evidenced the top expression outcomes throughout various periods of ED, SP, and GS (Figure S1)” – albeit PaPIP1.2 showed a high expression in the sampled material, they are no top. They are in line with other aquaporins of the cluster 1.
Response:
We thank the reviewer for this question. On lines 256-259 of revised MS, we have written the following:
“In our previous study, we used transcriptome sequencing to analyze the expression of PaAQP in the dormancy and sprouting periods of P. armeniaca, and PaPIP1-2 showed the relatively high expression levels throughout the various stages of ED, SP, and GS (Figure S1).”
Question: Why do you have this here and not in the objectives if is a sequential work??
Response:
We would like to propose that PaPIP1-2 gene has high expression in various periods of ED, SP, and GS, in order to elucidate its function involved in dormancy and germinate in the next step.
Question: There is no figures2
Response:
We apologize for this oversight. figure s2 was in lines 537-547 of revised MS.
Figure S2. Phylogenetic relationships analyses of PIP subfamily of AQP genes among the P. armeniaca genomes.
Question: There are no supplemental tables!!
Response:
We apologize for this oversight. We have reuploaded the supplemental tables.
Question: Line 309: “…and can accelerate the development of freezing tolerance [27].” - What this even mean?
Response:
We thank the reviewer for this question. On lines 323-325 of revised MS, we have written the following:
“Additionally, AT2G20050, which is homologous to protein phosphatase 2C, has been found to be downregulated in Arabidopsis, leading to strengthen the freezing resistance in transgenic plants [27].”
Question: Line 307 to 314: This is for the discussion section not result section.
Response:
We thank the reviewer for this question. On lines 328-330 of revised MS, we have written the following:
“This network analysis provides valuable insights into the functional associations of PaPIP1-2 and its potential role in plant responses to environmental stresses, particularly in cold conditions.”
Question: Line 328: “regulation of experience-dependent behavioral modifications in mice [30].” – Is this relevant?
Response:
We thank the reviewer for this question. Protein tyrosine kinase (PTK) was one of 6 genes and may be associated with cold tolerance (Table S5). On lines 339-341 of revised MS, we have written the following:
“Protein tyrosine kinase may tyrosine phosphorylation of SIRPα, which was shown to be induced by low temperatures [30].”
Question: Line 343: “Notably, all six gene pairs showed highly consistent expression patterns with PaPIP1-2 across all four evaluated phases.” – notably the expression pattern of these genes is not so highly consistent with PaPIP1.2 expression. Other exercise that is important is to analyze if the expression of those genes is high or low because if you have a similar expression pattern but with near 0 expression the gene will not have importance.
Response:
We thank the reviewer for this question. On lines 356-362 of revised MS, we have written the following:
“Remarkably, only PaPP2C displayed highly consistent expression patterns with PaPIP1-2 across all four evaluated phases. Other gene pairs include PaPTK and PaPIP1-2, PaFAD and PaPIP1-2, PaEREBP and PaPIP1-2, PaADH and PaPIP1-2, and PaZFER and PaPIP1-2 showed roughly similar expression trends with PaPIP1-2 across all four evaluated phases (The difference is that the expression of PaPTK, PaFAD, PaEREBP, PaADH, and PaZFER genes have decreased in the ED phase).”
Question: Other exercise that is important is to analyze if the expression of those genes is high or low because if you have a similar expression pattern but with near 0 expression the gene will not have importance.
Response:
We thank the reviewer for this question. Transcript levels of these 6 genes (including PaPTK, PaFAD, PaPP2C, PaEREBP, PaADH, and PaZFER) involved in cold resistance were detected in flower buds and stem during the PD stage, ED stage, SP stage, and GS stage of P. armeniaca (Figure 5b). Only the expression of PaZFER, PaEREBP and PaADH in ED stage, and the expression of PaADH in ED stage and GS stage in flower buds was relative lower than in other stages. So these genes probably plays an important role in dormancy and germination periods of P. armeniaca.
Question: I cannot understand the relevance of performing a -20 ºC freeze tolerance assay in yeast. Why did you studied the other PIPs?
Response:
Because there have been reports in the literature involving perform a -20 ºC freeze tolerance assay in yeast, we overexpressed several PIPs such as PaPIP1-1, PaPIP1-2, and PaPIP1-3 in yeast, and found that PaPIP1-2 increases cold accumulation and tolerance in yeast (Figure 6). Then PaPIP1-2 was choosen to overexpressed in Arabidopsis and the function of this gene was further analyzed.
The relevant references include,
Liu, Y.; Xie, L.; Liang, X.; Zhang, S. CpLEA5, the Late Embryogenesis Abundant protein gene
from Chimonanthus praecox, possesses low temperature and osmotic resistances in Prokaryote
and Eukaryotes. International Journal of Molecular Sciences 2015, 16, 26978-26990.
Question: Line 360: “…after exposure to -20°C had a noticeably enhanced growth compared to cells that had undergone…” – I cannot agree with this sentence. In the solid medium is not “noticeably enhanced”.
Response:
We thank the reviewer for this question. When visible to the naked eye, there are 4 obvious yeast spots in yeast strains overexpressing PaPIP1-2 in response to cold stress, while only 2 obvious yeast spots in yeast strains overexpressing PaPIP1-1 (red box in the following image), PaPIP1-3 and empty vector pGAPZA. This experiment demonstrated that yeast strains overexpressing PaPIP1-2 exhibited increased cold resistance of protein enrichment in yeast.
Question: Again, in the Arabidopsis tolerance assays the authors say that the results are evident when they are not. And these critical results are not quantified, they are an “evidence” that the plants “showed”. No quantification of nothing.
Response:
After a 10-day treatment period, the wild-type plants displayed yellow leaves, signifying their susceptibility to low-temperature stress, whereas the PaPIP1-2-overexpressing (OE) Arabidopsis plants exhibited enhanced growth and resilience to low-temperature stress (Figure 7a), which was accompanied by a reduction in MDA, an increase in proline accumulation and superoxide dismutase (SOD) activities, and cold resistance genes such as AtPUB26, AtBTF3L, AtEBF1-1, and AtRAV1, and a decrease in the ion leakage content compared with those of the WT plants (Figure 7c-i). We think these quantified and critical results can demonstrated that PaPIP1-2 gene improves cold stress tolerance in the P. armeniaca.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsReview Forests 2428763
Aquaporins play a major role in plant water uptake at both optimal and environmentally stressed conditions. Their influence on the cold resistance of Prunus armeniaca flower buds is of particular interest, since spring frosts are one of the main factors in reducing the yield of apricots. In the manuscript submitted for publication in “Forests Li et al focused on understanding the role of PaPIP1-2, a member of plasma membrane intrinsic protein subfamily of aquaporins in adapting to cold. The authors showed that PaPIP1-2 is essential in maintaining morphological growth recovery of flower buds at low temperatures. PaPIP1-2 exhibited co-expression with six genes implicated in low-temperature resistance at all stages of flower bud development. The function of PaPIP1-2 in cold tolerance was also confirmed using a yeast expression system and overexpression of PaPIP1-2 in Arabidopsis, as well as by assessing physiological parameters.
Overall, the authors presented convincing results confirming the engagement of aquaporins in cold acclimation of apricot flower buds. However, I have some minor concerns. In particular,
Line 21 “remained according to all stages” should be changed to” persisted at all stages”
Lines 118-119 “linked according to 5' extreme” to” ligated to 5'end”
Line 137 “random hexamer initiators” to “random hexamer primers”
Line 142 “Pieces” to “fragments”
Lines 159.160 “Scales” to” sepals”
Line 174 Please, decipher PaElf
Line 336 “were exhibited” should be changed to “exhibited”
Line 338 “6 protein” to “6 proteins”
Line 341 “We* to “we”
351 “to those of PaElf.” to “that of PaElf” or “to PaElf.””
Line 372 “cold accumulation” to cold acclimation
Lines 391-392 Fig7 legend (b) should be improved by inserting the name of the gene under consideration
Line 432” the maize PIP gene ZmPIP1-2 can be localized both in the plasma membrane and in the endoplasmic reticulum” to ”the expression of the maize PIP gene ZmPIP1-2 can be localized both in the plasma membrane and in the endoplasmic reticulum”
Line 445 “the amount of mRNA of PaPIP1-2 were found to be high” to” the amount of mRNA of PaPIP1-2 was found to be high”
Though I have mentioned some of the errors in the minor issues, I would recommend extensive language editing
Comments on the Quality of English LanguageI would recommend language editing
Author Response
Reply to the reviewer
Dear Editor of Forests,
Our original manuscript, titled “ Expression and functional analysis of the PaPIP1-2 gene during dormancy and germination periods of kernel-using apricot (Prunus armeniaca L.)” (Manuscript ID: 2428763), has been assessed by reviewers of Forests. The paper was revised according to your and the three reviewer’s suggestions. Extensive editing of the English language was performed by a native English speaker. We hope that our revised manuscript now meets the requirements for
publication in Forests.
Here, we resubmit our revised manuscript for publication in Forests. The revisions are listed below in detail. Important modifications are marked in red in revised manuscript.
Response to reviewer 2
Question: Line 21 “remained according to all stages” should be changed to” persisted at all stages”
Response:
We thank the reviewer for this question. On lines 24-27 of revised MS, we have written the following:
“The mRNA abundance of each PaAQP, which displayed coexpression with genes associated with resistance against low-temperature stress, remained consistent throughout all stages of P. armeniaca flower bud development.”
Question: Lines 118-119 “linked according to 5' extreme” to” ligated to 5' end”
Response:
On lines 24-27 of revised MS, we have written the following:
“The fusion construct was created by linking the coding regions of PaPIP1-2 cDNA to the 5' end of the GFP DNA.”
Question: Line 137 “random hexamer initiators” to “random hexamer primers”
Response:
On lines 156 of revised MS, we have written the following:
“Next, the first-strand cDNA was generated using random hexamer primers.”
Question: Line 142 “Pieces” to “fragments”
Response:
On lines 159-161 of revised MS, we have written the following:
“Small DNA fragments were then purified using the QIAquick PCR Purification Kit from Qiagen (USA), followed by end repair and poly(A) incorporation before ligation to sequencing adapters.”
Question: Lines 159.160 “Scales” to” sepals”
Response:
On lines 177-178 of revised MS, we have written the following:
“During the SP time, the flower buds were not enclosed by sepals, and the petals were not visible.”
Question: Line 174 Please, decipher PaElf
We thank the reviewer for this question. PaElf gene was elongation factor-1α. On lines 195 of revised MS, we have written the following:
“PaElf (elongation factor-1α) was used as the internal reference in the quantitative PCR.”
Line 336 “were exhibited” should be changed to “exhibited”
Response:
On lines 346-350 of revised MS, we have written the following:
“In this study, six genes, namely, protein tyrosine kinase (PaPTK), zinc finger protein (PaZFER), fatty acid desaturase family gene (PaFAD), protein phosphatase 2C (PaPP2C), ethylene-responsive element-binding protein (PaEREBP), and alcohol dehydrogenase protein (PaADH), exhibited robust coexpression with PaPIP1-2 in various tissues.”
Question: Line 338 “6 protein” to “6 proteins”
Response:
On lines 350-352 of revised MS, we have written the following:
“This interaction suggests the potential involvement of these six proteins in conferring low temperature invulnerability to P. armeniaca.”
Question: Line 341 “We* to “we”
Response:
On lines 353-358 of revised MS, we have written the following:
“To further analyze the network regulated by key PaPIP1-2, we examined the expression levels of the six genes associated with cold resistance within the coexpression network of PaPIP1-2.”
Question: 351 “to those of PaElf.” to “that of PaElf” or “to PaElf.”
Response:
On lines 553-554 of revised MS, we have written the following:
“The expression levels of these 6 genes are normalized to that of PaElf (elongation factor-1α).”
Question: Line 372 “cold accumulation” to cold acclimation
Response:
On lines 558-559 of revised MS, we have written the following:
“Figure 6 illustrates the effects of PaPIP1-2 overexpression on cold acclimation and tolerance in yeast. ”
Question: Lines 391-392 Fig7 legend (b) should be improved by inserting the name of the gene under consideration
Response:
We added PaPIP1-2 in the vertical axis of Fig7 (b). Also, the expression levels of cold resistance genes AtPUB26 (f), AtBTF3L (g), AtEBF1-1 (h), and AtRAV1 (i) were analyzed in Fig7.
Question: Line 432” the maize PIP gene ZmPIP1-2 can be localized both in the plasma membrane and in the endoplasmic reticulum” to ”the expression of the maize PIP gene ZmPIP1-2 can be localized both in the plasma membrane and in the endoplasmic reticulum”
Response:
On lines 432-434 of revised MS, we have written the following:
“However, Chaumont et al. (2000) [37] reported that the expression of maize PIP gene ZmPIP1-2 can be localized both in the plasma membrane and the endoplasmic reticulum.”
Question: Line 445 “the amount of mRNA of PaPIP1-2 were found to be high” to” the amount of mRNA of PaPIP1-2 was found to be high”
On lines 447-448 of revised MS, we have written the following:
“Particularly, during the sprouting (SP) period, the mRNA levels of PaPIP1-2 was found to be high, ”
Question: Though I have mentioned some of the errors in the minor issues, I would recommend extensive language editing
Response:
Extensive editing of the English language was performed by a native English speaker.
Reviewer 3 Report
Comments and Suggestions for AuthorsWith the widespread use of NGS analysis, genomes and gene sequences of various plant species have been revealed. A wide variety of plants are used in human life and a number of plants play important roles in their respective regions. Understanding genome sequences and gene expression patterns will expand the availability of plants and enrich human life.
In this paper, the authors focused on PaPIP1-2, an aquaporin of Prunus armeniaca L., based on RNA-Seq analysis. They suggested that PaPIP1-2 is involved in low-temperature tolerance in plants by performing subcellular localisation analysis, co-expression analysis and validation of low-temperature tolerance imposition using yeast. Furthermore, the authors tested whether overexpression of PaPIP1-2 enhanced low-temperature tolerance in Arabidopsis thaliana and showed that overexpression of PaPIP1-2 indeed enhanced low-temperature tolerance, enhancing SOD activity and proline accumulation during cold treatment, while reducing lipid peroxide accumulation.
This study, by successfully utilising RNA-seq information, identifies and analyses the function of important genes in a plant species for which information and resources need to be better developed, and could be an achievement that expands the potential use of Prunus armeniaca L.
Comments are as follows.
1. the reasons that led to the focus on PaPIP1-2 are important; modify Figure S1 to become Figure 1 and highlight the reasons for the focus on PaPIP1-2 in the text
2. this paper has been analysed using molecular biological methods, but some words appear to be infrequently used in this field. The problem could be solved by using an English language editing service where staff with expertise in molecular biology are present.
Comments on the Quality of English LanguageThis paper has been analysed using molecular biological methods, but some words appear to be infrequently used in this field. The problem could be solved by using an English language editing service where staff with expertise in molecular biology are present.
Author Response
Reply to the reviewer
Dear Editor of Forests,
Our original manuscript, titled “ Expression and functional analysis of the PaPIP1-2 gene during dormancy and germination periods of kernel-using apricot (Prunus armeniaca L.)” (Manuscript ID: 2428763), has been assessed by reviewers of Forests. The paper was revised according to your and the three reviewer’s suggestions. Extensive editing of the English language was performed by a native English speaker. We hope that our revised manuscript now meets the requirements for
publication in Forests.
Here, we resubmit our revised manuscript for publication in Forests. The revisions are listed below in detail. Important modifications are marked in red in revised manuscript.
Response to reviewer 3
Question: the reasons that led to the focus on PaPIP1-2 are important; modify Figure S1 to become Figure 1 and highlight the reasons for the focus on PaPIP1-2 in the text
Response:
We thank the reviewer for this question. There are several reason justify the need to study the function of PIP1.2. Firstly, Previous research has demonstrated the important roles of PIP subfamily members in cold acclimation and freezing tolerance. There are some connections between plant cold resistance and dormancy and germination (Li et al., 2020). PaPIP1-2 showed the differential expression in the dormancy and sprouting periods of P. armeniaca, this indicated that PaPIP1-2 may involved and play an important role in dormancy and germination periods of flower buds (Figure 1, Figure S1). Secondly, some typical protein domains exist in the PaPIP1-2 protein, such as MIP domain, Crp-type HTH domain, etc, this showed that PaPIP1-2 protein is potentially involved in crucial biological processes including signal transduction, transcriptional activation, and material transport, and need further research (Figure 2). Lastly, we found that several important proteins such as phosphatase 2C, Leucine-rich receptor-like protein kinase, which involved in cold resistance or germination speed, were potential interacting protein of PaPIP1-2 as confirmed by constructing a protein-protein interaction network. We are curious about the role of PaPIP1-2 gene in plant dormancy and germination, and resistance to cold stress. So other experiments such as transgenic and physiological analysis were taken to prove the function of this gene.
Related References
Li, S.; Wang, L.; Zhang, Y.; Zhu, G.; Zhu, X.; Xia, Y.; Li, J.; Gao, X.; Wang, S.; Zhang, J.; et al. Genome-Wide identification and function of aquaporin genes during dormancy and sprouting periods of Kernel-Using Apricot (Prunus armeniaca L.). Front Plant Sci 2021, 12, 690040.
On lines 79-83 of revised MS, we have written the following sentence involving reasons for the focus on PaPIP1-2 in the text.
“Among PIP subfamily genes, Arabidopsis homologous gene PIP1-2 showed the differential expression in the dormancy and sprouting periods of P. armeniaca, this indicated that PaPIP1-2 may involved and play an important role in dormancy and germination periods of flower buds.”
The question of modify Figure S1 to become Figure 1, because of this Figure S1 is not the first time appeared in this manuscript. This Figure was reproduced with permission from Shaofeng Li, Frontiers in Plant Science/Genome-Wide Identification and Function of Aquaporin Genes During Dormancy and Sprouting Periods of Kernel-Using Apricot (Prunus armeniaca L.). Considering the issue of repeatability, it can only be placed in the attachment. We thank the reviewer for this question.
Question: this paper has been analysed using molecular biological methods, but some words appear to be infrequently used in this field. The problem could be solved by using an English language editing service where staff with expertise in molecular biology are present.
Response:
Extensive editing of the English language was performed by a native English speaker.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsDear authors.
The critical results of the paper is exactly the same as I refuted in the previous review. So, the paper was not improved and I cannot accept it. As you say in the yeast results section, if you have a difference in 2 colonies at the last dilution that can be caused by normal pipetting erros, is not a strong and evident result. With the Arabidopsis plants is the same, I cannot concluded the same as you with your photos. And you ignored my request to functionally characterize the aquaporin.
Best regards.
Comments on the Quality of English LanguageThe english was barely improved.
Author Response
Reply to the reviewer
Dear Editor of Forests,
Our original manuscript, titled “ Expression and functional analysis of the PaPIP1-2 gene during dormancy and germination periods of kernel-using apricot (Prunus armeniaca L.)” (Manuscript ID: 2428763), has been assessed by reviewers of Forests. The paper was revised according to your and other reviewer’s suggestions. Thank you very much for the reviewers' excellent questions, and we have responded carefully. Extensive editing of the English language was performed by a native English speaker, and certificate of english editing was provided in submission system（Upload in Non-published Material）or email. We hope that our revised manuscript now meets the requirements for publication in Forests.
Here, we resubmit our revised manuscript for publication in Forests. The revisions are listed below in detail. Important modifications are marked in red in revised manuscript.
Response to reviewer 1
Question 1: “As you say in the yeast results section, if you have a difference in 2 colonies at the last dilution that can be caused by normal pipetting erros, is not a strong and evident result. “
Response:
Thank you very much for this question. Our yeast transformation experiment showed that there are 4 obvious yeast spots in yeast strains overexpressing PaPIP1-2 in response to cold stress, while only 2 obvious yeast spots in yeast strains overexpressing PaPIP1-1, PaPIP1-3 and empty vector pGAPZA (red box in the following image). A small volume (4 µL) of both the original (100, yeast strains overexpressing PaPIP1-2, PaPIP1-1, PaPIP1-3 and empty vector pGAPZA) and diluted yeast culture (10-1, 10-2, and 10-3, yeast strains overexpressing PaPIP1-2, PaPIP1-1, PaPIP1-3 and empty vector pGAPZA) was spotted onto solid SC-U medium containing galactose (2%). After a 48-hour incubation period at 30°C, the growth of transformed cells was observed and documented. We confirm that the volume (4 µL) of yeast solution in different carrier forms (yeast strains overexpressing PaPIP1-2, PaPIP1-1, PaPIP1-3 and empty vector pGAPZA) is consistent, and the temperature and time of cultivation are consistent. This is a true experimental result, and we confirm that it was not due to negligence in the experiment, such as errors caused by pipetting. The complementary evidence is after the application of low temperature strain, the average OD values were observed as 1.99 in the empty vector strains and 2.54 in the PaPIP1-2 strains, and the OD values measurement results are also real data (Figure 6b). This OD values can confirm the authenticity and validity of the results, which was the yeast colonies carrying the PaPIP1-2 gene exhibited more favorable growth compared to those carrying the empty vector strains under cold stress conditions (Figure 6a). Based on the significant cold resistance effect of PaPIP1-2 on yeast and Arabidopsis, we have applied for a national invention patent in China. Our research group has established a system for yeast transformation, Arabidopsis transformation, and functional identification. In the previous study of cold resistance of AQP homologous genes, we found that the colonies of yeast cells harboring the PaPIP1-3 and PaTIP1-1 genes were larger than those harboring empty vectors strains (Figure 8a of Li et al (2021)). After the -20 ℃ low-temperature treatment, the mean OD values were 1.99 vs. 2.54 and 2.48 (Figure 8b of Li et al (2021)). These results suggested that the PaPIP1-3 and PaTIP1-1 genes improved the low-temperature stress resistance of yeast cells (Li et al 2021, Front Plant Sci). this work was published in Front Plant Sci in 2021, and the first author of the published articles was the one submitted the new research work of PaPIP1-2 to Forests. So our experiment also proves that PaPIP1-3 homolougus gene PaPIP1-2, also has cold resistance in yeast and Arabidopsis (Figure 6, Figure 7 of this paper). Thanks.
Related References
Li, S.; Wang, L.; Zhang, Y.; Zhu, G.; Zhu, X.; Xia, Y.; Li, J.; Gao, X.; Wang, S.; Zhang, J.; et al. Genome-Wide identification and function of aquaporin genes during dormancy and sprouting periods of Kernel-Using Apricot (Prunus armeniaca L.). Front Plant Sci 2021, 12, 690040.
Related Figures
Figure 8 of Li et al (2021), published in Front Plant Sci.
Question 2: functionally characterize the aquaporin
Response:
We conducted a series of experiments to elucidate the function of the PaPIP1-2 gene.
Arabidopsis homologous gene PIP1-2 showed the differential expression in the dormancy and sprouting periods of P. armeniaca, this indicated that PaPIP1-2 may involved and play an important role in dormancy and germination periods of flower buds. Intracellular positioning of GFP-tagged PaPIP1-2 indicated that the modified proteins were predominantly located at the cell membrane. Through a PaPIP1-2 gene network analysis, we found that it exhibited coexpression with six genes related to low-temperature stress.Yeast strains overexpressing PaPIP1-2 demonstrated increased cold resistance, with enriched protein content in the yeast cells. After a 10-day treatment period, the wild-type Arabidopsis plants displayed yellow leaves, signifying their susceptibility to low-temperature stress, whereas the PaPIP1-2-overexpressing (OE) Arabidopsis plants exhibited enhanced growth and resilience to low-temperature stress (Figure 7a), which was accompanied by a reduction in MDA, an increase in proline accumulation and superoxide dismutase (SOD) activities, and cold resistance genes such as AtPUB26, AtBTF3L, AtEBF1-1, and AtRAV1, and a decrease in the ion leakage content compared with those of the WT plants (Figure 7c-i). In summary, our results highlight that the P. armeniaca PaPIP1-2 gene enhances cold stress tolerance and plays a crucial role in the dormancy and germination stages in P. armeniaca.
Thank you very much for these excellent questions. Thanks.
Author Response File: Author Response.pdf
