Dihydroisocoumarin Content and Phenotyping of Hydrangea macrophylla subsp. serrata Cultivars under Different Shading Regimes

Round 1

Reviewer 1 Report

The manuscript presents new interesting data on the comparison of the responses of three hortensia cultivars to three shading levels (no shade, partial shade, full shade). The results are clearly presented and discussed. However, some points should be corrected.

Abstract: There is no explanation of the abbreviation "PAR".

lines 121-123: Why wasn't the research design the same for both years?

Figure 1: It has been written "Hours of sunshine in 2018 (yellow) and 2019 (purple)" while Figure 1 is the grayscale graph. 

Lines 158-159: It has been written "In this study the last day of measurements (32 DALE) is taken as the plants response ...". Whereas, according to the data in lines 123-124, the experiment lasted 31 DALE in 2018 and 32 DALE in 2019. 

Table 1, footnote: It has been written "NIR = 7.80". Is this correct?

Subsections 2.3. Plant Phenotyping and 2.4. DHC Quantification: Were all parameters measured only in duplicate? 

Subsection Statistical analysis should be described in detail.

Conclusions should focus more on the obtained results than cite literature data.

References: There is double numbering.

 

 

Author Response

Dear Reviewer 1,

please find in the attached file our detailed response to your comments. We added in the PDF also the comments & answers to reviewer 2 and 3.

Regards,
Thorsten Kraska

(on behalf of all authors)

Author Response File: Author Response.pdf

Reviewer 2 Report

In the manuscript "Phenotyping of tea-hortensia cultivars under different shading regimes", the authors conducted an interesting and informative experiment comparing the effects of different light conditions and cultivars on biomass production and the content of dihydroisocoumarins and their derivatives. The authors concluded that cultivar was more important than light conditions for the production of dihydroisocoumarins.

The manuscript is generally well prepared and contains interesting data. However, I have some other important comments and some useful improvements that need to be made before a positive recommendation for publication. One important point that needs to be considered is the proposal of a new name for H. macrophylla subsp. serrata. In my opinion, the introduction of such a new name is not appropriate. I propose to use the Latin name throughout the manuscript instead of the name tea-hortensis. This could only be mentioned or somehow introduced in the final part.

 

There are several comments/suggestions for the manuscript as below:
- Title: I recommend using a Latin name instead of "tea-hortensia". The same applies to the entire text. Therefore, also adjust the text to reflect the new use of the name.
- Be precise in spelling the authors' last names (Wiesbaum or Wiessbaum?).
Abstract: If you have introduced an abbreviation (PD, HG), use it when necessary (lines 25 and 26).
Introduction:
- Line 41: To distinguish Hydrangea macrophylla subsp. serrata...
- Lines 44, 59, 60: Please add the appropriate reference.
- Line 75: English name of this species?
Materials and Methods:
- Explain the abbreviations (DOY...).
- Figure 1: There are no colors (yellow and purple), only gray and black. Please rewrite the figure legend. Also add the explanation of the x-axis.
- Line 142: Please refer to the appropriate figure.
- Line 145: Units for PAR?! PAR is usually measured as Photosynthetic Photon Flux Density (PPFD), with units in μmol/m^-2s^-1 (micromoles per square meter per second).
- Line 149: How were five plants randomly selected?
- Line 158: The dot is missing.
- Table 1: There is an error "Carotinoids", correct is carotenoids.
- Line 169: DHC quantification - Quantification was performed by what method? Please provide the appropriate reference.
- Please provide details of the model, company and country of all equipment used in the study (line 175, 176).

- The section on statistical analysis is missing. Please provide the statistical analysis methods used in your study and explain how many factors (with levels) were included in the individual ANOVA model for a particular parameter. Please also provide a copy of the table ANOVA showing the significance (with p-values) of the factors or interactions tested for a particular parameter.
Results and Discussion:
- Line 185: Global radiation, Line 188: Growth degree analysis, Line 198: The yield of flowers and buds - add this in the Materials and Methods section and explain.
- Line 199: Why did you study the effect of leaf age only in 2018?
- Line 203: More pronounced? The difference could only be significant or not significant.
- Line 209: ... Compared to older leaves... - Please refer to the relevant figure (Figure 4?). The same in line 211.
- Line 209: ... contained higher percentages of HG in young leaves compared to older ones - not for all shading scenarios! Please be precise when reporting results.
- Line 2015 and 2016: GH? Probably an error.
- Figure 4: It is very difficult to follow this figure. How many factors were included in the statistical analysis for a given parameter? Please indicate in the statistical analysis section.
- Figure 4E and 4F: There are no colors, please color the figure. I assume that the columns in Figures 4E and 4F represent the cultivars?
- Figure 4A, B, C, D: was only the leaf position factor significant here? What about the differences between cultivars?
- Line 226: there is no "d" letter in figure 4.
- Line 228: "...containing the highest content of PD in leaf dry matter" - not for the treatment "no shade". Also in both years?
- Figure 5A and 5B: If significant differences in hydrangenol content in leaf dry matter were only observed between the three cultivars or year, why are you presenting results and statistical differences for light conditions?
- Line 246: Please add standard error values if you are reporting means.
- Line 247: Add means and standard error values for HG in 2019 and 2018.
- Line 247: "... when pooled over all treatments" Was the interaction significant?
- Line 283, 284: dekegulac or dikegulac?
- Line 294: please add appropriate reference.
- Figure 7: This figure is also difficult to understand. Please provide the table ANOVA showing the significance (with p-values) of the factors or interactions tested for the hue parameter. The differences within the light regime were likely independent of cultivar?
- Line 354: It is difficult to see significant differences on figures, could you add these?
- The discussion on DHC content should be extended a bit.
- References: The current reference style is not in line with journal style, please refer to the submission guidelines for authors

Author Response

Dear Reviewer 2,

please find in the attached file our detailed response to your comments. We added in the PDF also the comments & answers to reviewer 1 and 3. We added also the graphical abstract.

Regards,
Thorsten Kraska

(on behalf of all authors)

Author Response File: Author Response.pdf

Reviewer 3 Report

General comments:

The synthesis and content of DHCs in leaves are the most important performance of tea-hortensias. In the present study, the authors hypothesized that tea-hortensias might require shading for the increased DHC production, and investigated the effect of three types of shading (no, partial and full shading) on the content of DHCs of three tea-hortensia cultivars. They found that, although partial and full shading resulted in slightly higher biomass production than no shading, the shading-regimes had no or little effect on DHC contents in all cultivars and the content is cultivar-dependent. These observations were new and clear-cut. This reviewer thinks the paper has a merit to be deposited in an archive. The authors also investigated the effect of shading on the plant growth by comparing VIs and color changes, and suggested that no-shade condition caused less vital plants. But this finding is inconsistent with the results of DHC content. Therefore, this reviewer recommends to omit the experiments on plant phenotyping, by deleting corresponding parts in the text, and change the title as shown below.

Specific comments:

L2-3: Change the title 'Phenotyping of tea-hortensia … ' to  'Dihydroisocoumarin contents of tea-hortensia …'

L16: some hortensia species

L20: The response of tea-hortensias as ….

L81: biomass yield and that …

L90-107, L110-111: delete

L134:  Shaded area indicates …

L154-168: delete

L172-174: How many grams of leaves were used for extraction? Show the leaf weight used.

L173: Change 'fermented, Fermentation'  to 'extracted, Extraction'

L179-178: … consisting of acidified water (0.1% formic acid; A) and acetonitrile (B) …

L191-192: Change 'figure 1, figure 2' to 'Figure 1, Figure 2'. Also in many other places, change ‘figure’ and ‘Fig.’ to ‘Figure’.

L197:  younger leaves contain larger amount of PD

L198: buds had significantly higher contents (μg/gFW)

L199: the effect of leaf age

L201: Change 'phyllodulcin' to 'PD'

L202: Figure 4

L206: In addition to PD, hydrangenol (HG) was assayed …

L207: HG contents were

L214: Change the order of respective panels in Figure 4. Figure 4A and 4B should show the data for PD, and Figure 4C and 4D for HG.

L214: Pooling data cannot be understood. Need to explain.

L220: Does a set of three bars of the same brightness in Figure 4A-D mean independent three  assays? Need to explain.

     This reviewer cannot understand what the pooled values mean. Is this necessary?

     Change the panels. A, B phyllodulcin (PD), C, D hydrangenol (HG)

L223-224: Change 'light, darker, dark' to 'bright, light, dark'.

L228: … having the highest content of PD

L234: in mid-September.

L244: HG content

L249: PD content

L254: tea-hortensia

L276: tea-hortensias

L278: PD yields

L293-427: delete

L442-443: delete

(end)

Author Response

Dear Reviewer 3,

please find in the attached file our detailed response to your comments. We added in the PDF also the comments & answers to reviewer 1 and 2. We added also the graphical abstract.

We discuss in detail your suggestions about phenotyping data.

Regards,
Thorsten Kraska

(on behalf of all authors)

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Some of the revision comments were answered with success. Nevertheless, in my opinion, the manuscript still needs some improvements and further clarification to improve its quality.

Specific suggestions are listed below:

- The style of the manuscript still does not meet the requirements of Plants journal in some aspects, please refer to the submission guidelines for authors (some journal names are abbreviated, others are not).
- Introduction: I suggest replacing Mediterranean green algae with another horticultural species. A short paragraph (two sentences) describing the importance of light on the performance of horticultural crops would be necessary. The following references should be used: Sharma, Y., Singh, H., & Singh, S. (2018). Effect of light interception and penetration at different levels of fruit tree canopy on quality of peach. Current Science (00113891), 115(8); Vosnjak, M., Mrzlic, D., & Usenik, V. (2021). Summer pruning of sweet cherry: a way to control sugar content in different organs. Journal of the Science of Food and Agriculture; Reale, L., Nasini, L., Cerri, M., Regni, L., Ferranti, F., & Proietti, P. (2019). The influence of light on olive (Olea europaea L.) fruit development is cultivar dependent. Frontiers in plant science, 10, 385.
- The units for PAR are still not correct! PAR is usually measured as Photosynthetic Photon Flux Density (PPFD), with units in μmol/m-2 s-1 (micromoles per square meter per second). Please correct this in Figure 3 and also add the units in the Materials and Methods section where you describe the light measurements.
- Line 167: Göttingen
- DHC quantification - Was the method developed by you? If not, please provide the appropriate reference from which the method was used.
- Please explain how many factors (with levels) were included in the individual ANOVA model for a given parameter in the statistical analysis section.
- FIGURE 4: It is still very difficult to follow this figure, you say "and E, F pooled over all three H. macrophylla subsp. serrata cultivars (green: 'Oamacha', orange: 'Odoriko 264 Amacha', purple: 'Amagi Amacha')". However, figures E and F are only gray. Please explain what the columns in Figures 4E and 4F mean: shading scenarios pooled across all three cultivars? You must remember that the multivariate statistical analysis shows a significant effect of the cultivar * shading and cultivar * leaf age interaction! But next you say "E,F: Significant differences for leaf position, pooled for all cultivars" This should be stated beforehand.
- Figure 4E and 4F: is the y-axis PD or HC content or perhaps DHC content?
- Supplementary material 7: multivariate analysis should be shown for a specific observed parameter (HC, PD). Now it is not clear whether the analysis is for HC or PD.
- Figure 4: Leaf position and leaf age, this is confusing. In Supp. Material 7, only the leaf age is given. It would be much easier to understand if you could describe in the statistical analysis section how many and which factors (with levels) were included in the individual ANOVA model for a particular parameter studied.
- The multivariate analysis for Figures 5, 6 and 7 is missing in supplementals.

Author Response

Dear Reviewer 2,

thank you again for your time reviewing our manuscript. You will find our answers in the attached PDF file.

Because we do not receive a response by the other two reviewers until now only your comments are included.

 

On behalf of the authors

Regards,

Thorsten Kraska

Author Response File: Author Response.pdf