Rebuttal
Dear Editors of Dr. Günther Hahne and Qiao Zhao
Enclosed pleased find main corrections according the reviewers’ comments and attached the revised manuscript of our manuscript (ID PCR-May-11-0287-M) entitled “Dynamics of phytohormone and DNA methylation patterns changes during dormancy induction in strawberry (Fragaria × ananassa Duch.)”. Revised portion in the manuscript are marked in red.
Thank you very much for your consideration.
Sincerely yours,
Zhang Li
Answers tothe Reviewers’ comments
Referee: 1
It is well known Auxin is an important plant hormone involved in controlling of the dormancy. Could it be possible that low temperature act by modulating the Auxin content, and the Auxin responsiveness together during the activity–dormancy transition? It will be interesting to check the Auxin responsiveness of AUX/IAA genes expression.
Answer: Although we did not detect the expression of Aux/IAA genes, in this experiment, we checked the PIN1 gene expression. It is well known that PIN1 is a pivotal protein of polar auxin transport, which plays a role in maintaining the high concentration of auxin (Kramer 2004). In addition, we had detected the expression of NCED1 (9-cis-epoxycarotenoid dioxygenase) gene. In the ABA biosynthetic pathway, NCED is the key enzyme in ABA biosynthesis in higher plants (Iuchi et al. 2001). In the revised manuscript (page9, 10, 11), we have addedresults of the expression of both PIN1 gene and NCED1 gene.
Besides Auxin and ABA, Gibberellic acids (GAs) are also the important plant hormones involved in the timing of dormancy establishment and chilling induced release. Did the authors detect the change of the GA content or the expression of GA responsive genes?
Answer: In the pre-experiments, we also detected change of the GA3 content. However, the results of the experiment showed that the GA3 levels had changes without significance. Thus, we did not detect GAs content and also the expression of GAs responsive genes in the follow-up experiments.
In the introduction part, the objective of the research should be more preciseous.
Answer: According the Reviewer’s comments, we have revised the objective of the research in the introduction part (page 4).
-There are many grammar mistakes. Some sentences are not smooth. Please check and correct them carefully, make it more fluent, which would be easier to understand your description and point.
Answer: We have tried our best to revise the English of the whole manuscript carefully, and also have asked our colleague who has better level of English language to help us for checking the English of the manuscript.
-Figure 2, the detection of ABA and Auxin should be confirmed by mass spectrum.
Answer: Considering no condition using mass spectrum to detect hormones presently in our laboratory, the HPLC method for detecting the ABA and Auxin was used according the reported methods (Nagar 1995; Jinu and Byoung 2009; Zhang et al. 2008). In order to eliminate impact of the instruments and the operating conditions on the analysis result, so to improve the accuracy of results, the standard solution of IAA and ABA were used to detect the recovery rate according to their extraction and purification process. Moreover, we tested standard samples and drew standard curve before sample measurements.
Supplementary Fig. 1 Separation of standard IAA and ABA using HPLC (a). Separation of IAA and ABA in tested sample using HPLC (b)
-Figure 5, the lines connecting “E.Coli” “Homo Sapiens” to the phylogenetic tree are missing.
Answer: We have corrected it in Fig. 5 (page 21)
- The paper “Horvath et al 2009” does not exist. Is it “Horvath et al 2008” in the references?
Answer: Yes, the reviewer is right. We have checked and corrected“Horvath et al 2009” in “Horvath et al 2008” in the references.
Referee: 2
1, please double check the spelling and format
Answer: We have checked the spelling and format error, and also have asked our colleague who has better level of English language to help us for checking the English of the manuscript.
2, suggest change the figure labels from 8h to SD and 16 h to LD.
Answer: The figure labels have been changed from 8h to SD and 16 h to LD.
3, Figure 6 and 7: change “each dot” to “each data”
Answer: The words of “each dot”have been changed in “each data” in Figure 6 and 7 in the revised manuscript.
4, Figure 6: I understand the qRT-PCR data were normalized to FaGADPH gene’s expression level. The question is what happened to the transcriptional level to the housekeeping gene FaGADPH during the genome wide DNA-methylation? Did the author see any of the housekeeping gene changes because of the methylation? Please check the original data and discuss as mentioned in page9, line 42-49.
Answer: Expression of the housekeeping geneFaGAPDH was unchanged, because of the global DNA hypermethylation during dormancy induction in strawberry. The FaGAPDH gene with constant expression levels through all treatment stages (Supplementary Fig. 2) was used to normalize raw data and to calculate relative expression levels. The results has been discussed at (page11) in revised manuscript.
Supplementary Fig. 2Transcriptional analysis of GAPDH gene by qRT-PCR (a All Star; b Darselect). Cycle threshold (Ct) values represent the average of three technical replicates
5, please discuss the relationship of the temperature /photoperiod, hormone changes and DNA methylation. Do the environmental changes cause the DNA methylation and DNA methylation cause the hormone changes? Please discuss.
Answer:The results of the current experiment clearly showthat dormancy is a gradual process, which was induced by the interaction of both temperature and photoperiod factors, which trigger changes in the levels of phytohormones such as IAA and ABA and in the expression of the related genes. During the induction of strawberry dormancy, DNA methylation increased, which lagged behind changes in IAA levels but was synchronized with changes in ABA levels. Considering report of that ABA plays a pivotal role in seed development and dormancy by direct induction of histone ubiquitination, acetylation and methylation-dependent chromatin (Chinnusamy et al. 2008), it might be speculated that ABA and IAA regulate DNA methylation by an unknown mechanism in plant dormancy. DNA hypermethylation, in combination with slow-down metabolic, is likely to repress the growth-associated gene expression during induction of dormancy.
The issue had discussed (page 12) in revised manuscript.
6, Page9, line 50: the ABA and DNA methylation seems change earlier in the Deselect. But response later for IAA, please discuss this issue.
Answer: The response later for IAA seems have some contradiction to conclusion in discussion part. Thus, we removed some improper sentences and had corrected some statements in the revised manuscript (page11).
7, Page9, line 56: the author explained the cultivar effects but seems like conflict to the abstract part (see page 1, line 52), please clarify this.
Answer: When dormancy in strawberry plants were induced by environmental factors, the two tested cultivars have different responses in time with the different degree of cold tolerance and depth of dormancy. However, the inside responding materials, endogenous phytohormone and DNA methylation, were homologous in both cultivars during the induction of dormancy. This is indicated that dormancy induction was mostly caused by plant intrinsic signals, somehow regardless of the cultivars.
The conflict to the abstract part has beencorrectedin the revised manuscript.
8, Page 9, line 58: the author claimed that the All star cultivar is more tolerant to cold, please clarify this. Meanwhile, it will be nice if the authors can add a little of cultivar background/ explanations for choosing the two cultivar in the introduction part.
Answer: We have added the background of the two tested cultivars in the introduction of revised manuscript (page 4).