Ethylene-regulated immature fruit abscission is associated with higher expression of CoACO genes in Camellia oleifera

Immature fruit abscission is a key limiting factor in Camellia oleifera Abel. (C. oleifera) yield. Ethylene is considered to be an important phytohormone in regulating fruit abscission. However, the molecular mechanism of ethylene in regulating fruit abscission in C. oleifera has not yet been studied. Here, we found that the 1-aminocyclopropane-1-carboxylic acid (ACC) content was significantly increased in the abscission zones (AZs) of abnormal fruits (AF) which were about to abscise when compared with normal fruits (NF) in C. oleifera ‘Huashuo’. Furthermore, exogenous ethephon treatment stimulated fruit abscission. The cumulative rates of fruit abscission in ethephon-treated fruits (ETH-F) on the 4th (35.0%), 8th (48.7%) and 16th (57.7%) days after treatment (DAT) were significantly higher than the control. The ACC content and 1-aminocyclopropane-1-carboxylate oxidase (ACO) activity in AZs of ETH-F were also significantly increased when compared with NF on the 4th and 8th DAT. CoACO1 and CoACO2 were isolated in C. oleifera for the first time. The expressions of CoACO1 and CoACO2 were considerably upregulated in AZs of AF and ETH-F. This study suggested that ethylene played an important role in immature fruit abscission of C. oleifera and the two CoACOs were the critical genes involved in ethylene's regulatory role.

1) My main concern with the manuscript is that is rather lacking in novelty; the role of ethylene in fruit abscission is well described, and although the authors describe it in Camellia for the first time here, none of this is a surprise. If the authors added some new insight into the role of ethylene in fruit abscission, it would make this a much better manuscript.

2) Ethephon
The authors use ethephon, but never tell the reader what ethephon is, or does.
3) Sample size, statistics From the authors descriptions, it is impossible to tell whethera) each experiment was performed with three samples, and all data are presented in supplementary file 3 or b) each experiment was performed 3 times, and only the experimental means of each experiment are reported in supplementary file 3. This is a very big distinction, and needs clarifying. If each experiment was performed 3 times, what was the sample size of each experiment?
All data were analysed using ANOVA, but it seems unlikely that with n=3, that all data were normally distributed. Did the authors check this before performing ANOVA?
Decision letter (RSOS-202340.R0) We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Mrs Ma
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Please attempt to improve the grammar in the manuscript as requested by reviewer 2. In particular the grammar in the title 'Ethylene effects immature fruits abscission is associated with higher expression of CoACO genes in Camellia oleifer' should be checked.
Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) The following are minor corrections. 1. Please delete sheets 2 and 3 in the supplementary file 3 as sheet 1 includes all the information needed; 2. Please submit the CDS sequence of CoACO1 and CoACO2 to GenBank and provide the GenBank ID in the manuscript instead of putting the sequences in supplementary file 1; 3. In Figure 2, please highlight the Arabic alphabets and arrows with colors. 4. In Figure 3, please add 'A' at the first column diagram; 5. The GeneBank numbers showed in Fig. 4C are not the same as the ones in "Supplementary file 2". Please double check. 6. Numbers should be in academic writing, for example, 1057 bp in Table 1 should be 1,057 bp; 7. In Line 124, could you explain why the optimal ethephon concentration is 2 g/L? References might be needed here to show why this concentration is used; 8. Line 208, 'On 4th DAT' should be revised as "On the 4th DAT"; 9. In line 264, please explain a little bit why you choose ACO1, ACO2, and ACO3 from grape to the protein alignment with CoACO1 and CoACO2. Similarly, please also explain the reason for using full-length ACO protein sequences from Arabidopsis, grape, tomato, sorghum, and rice in the phylogenetic analysis; 10. Lines 595 and 604, 'Relative levels expression' and 'Relative levels of expression' should be revised as 'Relative expression levels'.
Reviewer: 2 Comments to the Author(s) In this manuscript, the authors investigate the role of ethylene in fruit abscission in Camellia olifera. This is an important subject, but I feel like the role of ethyene in this response is already well characterised -as the authors discuss in their introduction.
Major points: 1) My main concern with the manuscript is that is rather lacking in novelty; the role of ethylene in fruit abscission is well described, and although the authors describe it in Camellia for the first time here, none of this is a surprise. If the authors added some new insight into the role of ethylene in fruit abscission, it would make this a much better manuscript.

2) Ethephon
The authors use ethephon, but never tell the reader what ethephon is, or does.
3) Sample size, statistics From the authors descriptions, it is impossible to tell whethera) each experiment was performed with three samples, and all data are presented in supplementary file 3 or b) each experiment was performed 3 times, and only the experimental means of each experiment are reported in supplementary file 3. This is a very big distinction, and needs clarifying. If each experiment was performed 3 times, what was the sample size of each experiment?
All data were analysed using ANOVA, but it seems unlikely that with n=3, that all data were normally distributed. Did the authors check this before performing ANOVA?
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Decision letter (RSOS-202340.R1) We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.

Dear Mrs Ma,
It is a pleasure to accept your manuscript entitled "Ethylene-regulated immature fruit abscission is associated with higher expression of CoACO genes in Camellia oleifera" in its current form for publication in Royal Society Open Science. The comments of the reviewer(s) who reviewed your manuscript are included at the foot of this letter.
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal. Many thanks for reviewing our manuscript. All points you raised have been considered carefully and answered. The title "Ethylene effects immature fruits abscission is associated with higher expression of CoACO genes in Camellia oleifera" has been revised as "Ethylene-regulated immature fruit abscission is associated with higher expression of CoACO genes in Camellia oleifera". All line numbers of the revision referenced in the manuscript are from the revised manuscript (the highlighted copy). 3. In Figure 2, please highlight the Arabic alphabets and arrows with colors.
Answer: Thanks for your suggestion. We have highlighted the Arabic alphabets and arrows with colors in the revised manuscript (Fig. 2). Figure 3, please add 'A' at the first column diagram; Answer: We are sorry for our mistake. We have added 'A' at the first column diagram in Fig. 3 in the revised manuscript. Fig. 4C are not the same as the ones in "Supplementary file 2". Please double check.

The GeneBank numbers showed in
grape, tomato, sorghum, and rice in the phylogenetic analysis.
Answer: Thank you for this comment. The coding sequences of CoACO genes were used as a query for BLAST searchers to acquire the ACO genes that have been reported in other species. It was found that the ACO genes from Arabidopsis, grape, tomato, sorghum, and rice had a certain similarity with CoACO genes and therefore we used these ACO proteins for the phylogenetic analysis. The phylogenetic analysis showed that the amino acid sequence similarity between ACOs from grape and CoACOs were relatively high, so we chose ACO1, ACO2, and ACO3 from grape to the protein alignment with CoACO1 and CoACO2.
10. Lines 595 and 604, 'Relative levels expression' and 'Relative levels of expression' should be revised as 'Relative expression levels'.
Answer: We are sorry for our mistakes. "Relative levels expression" and "Relative levels of expression" have been revised as "Relative expression levels" in the revised manuscript (L.625, L.634).

Reviewer: 2
Point 1: My main concern with the manuscript is that is rather lacking in novelty; the role of ethylene in fruit abscission is well described, and although the authors describe it in Camellia for the first time here, none of this is a surprise. If the authors added some new insight into the role of ethylene in fruit abscission, it would make this a much better manuscript.
Answer: Thanks for your suggestion. Ethylene and abscisic acid are widely recognized as hormones that promote the abscission of plant organs. However, our previous experiments showed that there was no significant difference in abscisic acid content between abscission zone of normal fruit and that of abnormal fruit, while ethylene did. So we explored the important role of ethylene in the fruit abscission of Camellia oleifera in this study, and we are going to conduct an in-depth study on how ethylene regulates the fruit abscission of C. oleifera in the next stage.
The authors use ethephon, but never tell the reader what ethephon is, or does.
Answer: We are sorry for our incomplete statement. "Ethephon (2-chloroethylphosphate acid) is an ethylene-releasing molecule which is stable in a low pH solution, but it hydrolyses in the higher pH of plant tissues releasing ethylene (Ferrara et al., 2016). Ethephon is a gaseous plant growth regulator that can promote fruit ripening, affect fruit color, firmness, carbon dioxide production and other This is a very big distinction, and needs clarifying. If each experiment was performed 3 times, what was the sample size of each experiment? All data were analysed using ANOVA, but it seems unlikely that with n=3, that all data were normally distributed.
Did the authors check this before performing ANOVA.
Answer: We are sorry for our incomplete statement. Each experiment was performed with three samples and each sample has three technical replicates in our study. And all data presented in supplementary file 3 were obtained by taking the average of three biological and technical replicates. We have checked the data before performing the ANOVA.