Breaking Buds—Stop and Go? Acid Invertase Activities in Apple Leaf Buds during Dormancy Release until Bud Break
Round 1
Reviewer 1 Report
Dear authors,
I've read you work carefully and I'll revise it with a very critical viewpoint. I do not agree with the construction of the arguments based only on invertase activities. Invertase is involved in the process of bud break - that's described in literature - but many other variables are also involved. Inv activity by itself do not provide all the conclusions shown in this work. For instance, the conclusion 'cwINV activity increased during the observation period in both years. This indicates an increase in sink strength, enabling the buds to attract energy-rich molecules and, ultimately, promoting bud break.' That affirmative is true (although no data about sink strength have been collected in this work) but fundamental events like phytohormones are closed related and no evidence was presented about this.
Other conclusion was 'The sharp increase in vacINV activity during bud break suggests a role in the promotion of expansion growth and hence of bud break in apple leaf buds also for this enzyme.' Again, the authors do not show evidences for that. About this another conclusion 'However, vacINV seems to be more sensitive to low and freezing air temperatures before bud break, as it negatively responded to the cold stress induced by abrupt temperature drops during the observation period for our study.', I do not agree with it. We all know that low temperatures affect negatively the enzyme activity whatever it is, and consider that 'The purpose of this down-regulation of vacINV activity may be to indirectly protect the sensitive developing leaf tissues from exposure to cold by temporarily' sounded baseless.
From reasons considered above, I believe that this work should not considered for publication.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript "Breaking Buds – Stop and Go: Acid Invertase Activities in Apple Leaf Buds of the Cultivar Idared during Dormancy Release until Bud Break" is a paper reporting the results of original research, which falls within the scope of the journal Agronomy.
The subject of investigation is to evaluate activities of vacuolar invertase (vacINV) and cell wall-bound invertase (cwINV) in leaf buds from ecodormancy release to bud break under natural orchard conditions.
The authors collected buds of the apple cultivar Idared from fifty trees in Haidegg (Austria) over two consecutive years (2020 and 2021). They performed specific assays on physiologically active tissues of these buds to reveal vacINV and cwINV activities, according to previously published protocols.
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The work presented in this manuscript offers a comprehensive study of the activity of these two acid invertases. The paper is well structured and the writing style easy to understand.
The introduction is very clear and describes the context of the study in great detail, based on recent articles. The role and function of vacuolar invertase (vacINV) and cell wall bound invertase (cwINV) are well described. The results are presented with only one synthetic figure. The accompanying text is clear, although further explanations on some points could help readers (see specific comments).
However, the discussion section is more of a state of the art than a discussion. The results of the study are not sufficiently highlighted and not adequately compared with previous works. The results begin to be cited 80 lines after the start of the section. In addition, it would be nice to have a real comparison between the two years of data (temperatures and enzyme assays), which were described separately in the results section. The lack of a real balance between data obtained during the two years of the study minimises some results. By way of example, the difference in the amplitude of the mean temperatures between 2020 and 2021 is not shown. Therefore, the discussion section must be revisited before publication.
Specific comments
Previous studies have shown that acid invertase activities follow a circadian rhythm. This information does not appear in the article. Please include it in the introduction and discussion. Specify in the protocol whether or not all samples were collected taking into account the circadian rhythm.
Page 4, line 180: please specify that remaining tissues are physiologically active tissues
Page 4, line 183-185: the sentence "To express enzyme activity….dry weight of the buds" is inaccurate and must be modified. The authors need to know the mean dry weight of physiologically active tissues (without bud scales) in order to express enzyme activity on an adjusted dry weight [aDW] basis. They can obtain it from the respective initial dry weight (line181) and percentage of remaining tissues (line 182). Is this the method that was used?
Page 5 to 7: please modify the order of the paragraphs in the results section to follow the scientific approach (field then lab): 3.1.phenological Growth stages of Apple Leaf Buds-3.2. Air temperatures-3.3. Acid Invertase Activities in Apple Leaf Buds.
Page 6, line 289-290: The information "actual fluctuations in mean air temperatures" is not precise. Please indicate the reference period you considered. Please explain what allows you to say "the mean air temperature of the interval did not represent the actual fluctuations in mean air temperatures.". Did you carry out a statistical analysis to confirm that?
Page 6, line 291: please replace "2022" with "2021"
Page 7, line 298-300: The information "summarized the daily and nightly mean air temperatures during these periods" is not precise. Please, indicate the reference period you considered.
Page 9, line 334-335: please move this sentence to the Introduction section and change the reference to a more relevant one (e.g. FAO).
Page 9, line 337: please replace "Firstly" with "firstly"
Page 9, line 341-342: please review this sentence. It will be easier to understand if you simply state that your study is interested in the transition from ecodormancy to bud break.
Page 9, line 349: please cite one or more recent papers reviewing the main processes involved.
Page 9, line 404: please replace "Firstly" with "firstly"
Page 9, line 405: please replace "from dormancy" with "from ecodormancy"
Figure 1: please include in the title "mean" before "air temperature" and replace "state" with "stages". Use identical ordinate scales for 2020 and 2021 graphs. Check the distribution of the colours on the curves. The yellow and blue colours should appear every day. This is not the case, for example, for the period 81-86 in 2020 and 88-92 in 2021.
Comments for author File: Comments.pdf
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
The manuscript is considering the important physiological aspects of bud dormancy what is especially significant currently.
The climatic conditions (2.5.) should be corrected. This part is not incomprehensible and says nothing.
The Results should be more clearly described.
The subtitles are described as the year, but the results are only to the ca 102 days (?).
The Introduction and the discussion are too long. Both should focus on the aspects conncted with the work only.
I can't open and I can't see the additionally files.
It's correct.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Dear Authors, this version of the MS resubmitted presented significant improvements. In my last revision, I focused that the conclusions based on Inv activities were not adequate because your set of results were limited. But now I felt that you guys modified the way the results are interpreted as this sounds more suitable for publication in Agronomy.
