Orchard Planting Density and Tree Development Stage Affects Physiological Processes of Apple (Malus domestica Borkh.) Tree

Round 1

Reviewer 1 Report

The study presented here aimed to fill the current lack of observations on the change of plant photosynthetic capacity and pigments with leaf age and light availability. The authors conducted leaf gas exchange measurements to quantify the change of photosynthesis as well as leaf chemical analyses to study the change of pigments and starch. The results convincingly showed that photosynthetic capacity and pigments would respond to age and light. I find this study a welcoming addition to existing work if the authors can address a few issues listed below. 

The method section could use more details. Firstly, what is the plant height and crown radius? This information could be useful to interpret the meaning of distance. In fact, I’m surprised that there is no mention of leaf area index or canopy cover. Those metrics could be easily used as alternatives to plant distance and are more generalisable. 

Other details include the following. Some abbreviations, such as BBCH and HPLC are used without definition. The ambient environments are not described when the leaf gas exchange measurements were taken. This is a bit worrying because although the conditions in the chamber can be controlled. Single leaves may not respond to the chamber condition rapid enough. For example, 9am would mean very different light and temperature in early spring and mid summer. It is likely that light intensity is still low and temperature cool at 9am in early spring, resulting in the plants still ‘waking up’. Measurements of gas exchange during those two periods thus can be biased by ambient condition.  

There is no analysis of the relationship between photosynthesis rate (or better, capacity) and pigment concentrations. This could be helpful because this paper assumed the cause of decline in photosynthetic capacity with leaf age as a change in leaf chemical. 

Detailed commons

L10: It may be easier for the reader to understand the importance of this study if presented with a brief introduction of the background and initiative.

L19-L20: BBCH and season are used inconsistently. I would suggest simply use one and avoid the other.

L108-109: 20 measurements in total or each season? It’s unclear how the campaigns are scheduled.

Figure 1: Could the authors show raw data and color different distance by color? The current presentation makes me wonder whether distance has any impact at all. Same applies to figure 3 and 5.

Author Response

In the methods was improved providing the additional information: the measurements were performed in a fully matured orchard, which was planted in 2001. The information on crown height and diameter was added seeking to facilitate the understanding of fruit tree density.

BBCH - Biologische Bundesanstalt, Bundessortenamt and Chemical Industry (German scale used to identify the phenological development stages of a plant). For apple trees in May - leaves are fully expanded, BBCH 20-25; middle of July - beginning of apple maturity BBCH 73 – 75, August - harvest time BBCH 87-88.

For a better understanding of the measurement time, we provide measurements of the photosynthetic rate dynamics of the same experiment at each stage of development. We choose the measurement time based on Fleancu, 2007 and also based on our own dynamic measurements (Fig. 1).

Fleancu, M. (2007). Diurnal and seasonal course of the rate of the photosynthesis in the apple tree case in the conditions from the fruit-growing region Pitesti-Maracineni. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Horticulture, 64(1-2), 223-228

The relationship between photosynthesis rate, pigment concentrations and other measurements are presented in Figure 9.

L10: Thank you for your comment, the abstract was supplemented by additional information

L19-L20: corrected

L108-109: Measurements of photosynthesis were performed on 3 trees selecting 3 leaves The methods were corrected.

More detailed data for the assessment of seasonality and planting density are provided in the supplementary material in Tables S1, S2, S3.

Author Response File: Author Response.pdf

Reviewer 2 Report

The researchers evaluated the impact of planting density (four levels between 0.25 and 1 m) and developmental stage (3 levels from fully expanded leaves to maturity) on physiological and metabolic parameters of apple tree leaves. Specifically, they studied photosynthetic parameters (net photosynthesis, stomatal conductance, and transpiration rate), pigment content (chlorophylls and carotenoids), sugar content (fructose, glucose, and sorbitol), and starch content.

The Introduction section is quite informative and sufficiently describes the scope of the research. However, some clarifications are necessary.

Materials and methods require amendments, particularly in the experimental design which is not properly described. Especially the age of trees should be provided which will help to better evaluate the results and discussion sections as well.

Conclusions can be improved by providing take-home messages.

Specific comments are following.

• Line 27. Keywords should not be included in the title. Please remove or substitute “apple”
• Line 38. “Leaf”
• Light 39. Light is an environmental factor.
• Lines 39-40. Please rephrase this sentence because it is not clear.
• Lines 58-60. Please rephrase this sentence because it is not clear.
• Lines 73-75. This can be separated into two sentences for better consistency.
• Lines 85-89. I do not think this paragraph is relevant since you do not report findings about the impact of rootstock. Maybe you should only add a comment about P 22 when you mention it in materials and methods.
• Lines 94-97. How old were the trees in your trial and when did you plant them (also add it in the abstract)?

Please add any necessary details regarding the experiment. For example, did you use shading nets as you mentioned in the introduction?

• Line 97. Please rephrase this sentence because it is not clear. Add as many details as possible in order for the reader to fully understand the experimental design.
• Line 107. Please explain why you chose 1000 μmol m-2 s-1 as light intensity.
• Lines 151-152. I understand that Institute of Horticulture (LAMMC, Lithuania) is the area where the orchard is planted, correct? If yes, please specify that the meteorological station is close to the orchard.
• Line 154 - Table 1. These are mean values, correct? Please specify it in the caption. The same applies for Table 2.
• Line 168. 27% and 23% between summer and which treatment?
• Lines 186-187. These values are repetition from Table S3 and I suggest that you omit them. You may add percentages instead.
• Lines 187-188. Fru and Sor correspond to Figure 5A and 5C. Moreover, the figure shows that both sugars are lowest in mid-summer and not in summer. Please, also mention Glu results.
• Lines 251-252. Photoperiod is the length of the day. Please amend the sentence.
• Lines 257-258. Do you have a theory about this finding?
• Lines 259-260. I cannot comment on this statement since I do not know the age of the trees. Please, add this information in materials and methods.
• Lines 273-274. Probably due to non-excessive light intensity. Do you have any light intensity data from the meteorological station in order to support these findings?
• Lines 298-303. The conclusions are quite poor and repeat some of the results. Please provide take-home messages including your most important findings. For example, the gradual increase of some parameters with decreasing planting density and the impact of development stage in sugar content.

Author Response

We supplemented the methods with information about the age and size of trees, as well as we clarified the measurement methods.

• Line 27. Changed to malus domestica
• Line 39-40. Recommended changes have been made

• Lines 39-40. and Lines 58-60. Sentences improved.
• Lines 73-75. Corrected.
• Lines 85-89. The P22 is a super-dwarf rootstock, we wanted to briefly introduce the classification of rootstocks, the reader would be easier to understand that this is the lowest rootstock.
• Lines 94-97. In the methods, we provided information that the measurements were performed in a fully matured orchard, which was planted in 2001. We also supplemented the methods with information about crown height and diameter to facilitate better understanding of fruit tree density.
• We do not use shade nets, but when planting at high density, the trees shade each other. Therefore, in the introduction, we also discuss the results that researchers obtained using nets shading.
• Line 107. According Li-Cor protocols (2014)  1500 μmol m^–2 s^–1 is used for plants growing in sunlight, 500 μmol m^–2 s^–1 - for shaded plants, 1000 μmol m^–2 s^–1 - for half shaded plants. Idris et al. (2019) reasurements shown that starting from 900 μmol m^–2 s^–1 net assimilation is stable for plants growing in the sun and in the shade

Idris, A., Linatoc, A. C., Bakar, M. B. A., & Takai, Z. I. (2019, July). Effect of light intensity on the gas exchange characteristics and total pigment content of Psidium guajava. In IOP Conference Series: Earth and Environmental Science (Vol. 269, No. 1, p. 012020). IOP Publishing

Evans, J. R., & Santiago, L. S. (2014). PrometheusWiki gold leaf protocol: gas exchange using Li-COR 6400. Functional Plant Biology, 41(3), 223-226

• Lines 151-152. Yes, you understood correctly. The experimental orchards are located in the Institute of Horticulture, LAMMC in the same place there is also a meteorological station from which the data were collected. We supplemented the methods with this information
• Line 154 - Table 1 is average values of monthly temperature and table 2 total monthly precipitation. We specified it.
• Line 168.  Effect of seasonality regardless distances between trees is demonstrated in Fig. 1, and 27% and 23% between summer is in average of treatments.
• Lines 187-188. corrected
• Lines 251-252. The sentence was amended.
• Lines 257-258. Increasing distances reduce competition for light, water and nutrients between fruiting trees, which can be one of the main reasons for increase of photosynthetic rate, stomatal conductance and transpiration rate.

Lines 259-260. In the methods was improved providing the additional information: the measurements were performed in a fully matured orchard, which was planted in 2001. The information on crown height and diameter was added seeking to facilitate the understanding of fruit tree density.

• In the methods was improved providing the additional information: the measurements were performed in a fully matured orchard, which was planted in 2001. The information on crown height and diameter was added seeking to facilitate the understanding of fruit tree density.

• Lines 273-274. We do not have data on sunlight intensity yet, but in future we will measure it.
• Lines 298-303. The conclusions are quite poor and repeat some of the results. Please provide take-home messages including your most important findings. For example, the gradual increase of some parameters with decreasing planting density and the impact of development stage in sugar content. – Conclusions was improved

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear authors,

The manuscript “Orchard planting density and tree development stage 2 affects physiological processes of apple tree” is relevant for the Journal of Agronomy. The methods used during this study are common and sound, and conclusions are supported by the data provided. However, the manuscript has many weak points and cannot be considered suitable for publication in this form.

As first, the research lacks novelty, since neither novel findings from large datasets are presented, nor new analytical techniques are demonstrated. Additionally, on material and methods important information is missing. While reviewing the manuscript several questions have been raised, for instance:

• What was the size of the study area?
• In how many rows trees were planted?
• how many trees were planted per treatment?
• When did measurements take place?
• How many days of measurements per studied month (May, July, August)?
• When you refer to meteorological conditions you compare temperature mean monthly values in 2018 and 2019 to multi-annual. Is that a 100-year average? Please clarify.
• In the result section, you refer to seasonal changes. I would suggest that it is more appropriate to refer to monthly changes or development stage changes.
• In line 165, the decrease you found in photosynthesis, stomatal conductance, and transpiration rate, is relevant to May’s values?
• In line 168, by summer readings do you refer to July readings?

Furthermore, some clarifications are essential on the experimental design. Please, provide more details.

Low photosynthetic rates sometimes are controlled by low nighttime temperatures, accompanied by short photoperiod (below 12h). If you have access in such data, it might be interesting to explore the effect of these parameters in your measurements.

Taking all together I think the manuscript needs major revision before acceptance.

Author Response

• 15 single trees were fully randomized. The experiment was arranged in complete randomization in two rows which were not edge rows.
• All measurements were made on the same day (per studied month, at specific BBCH-scale development stage), with windless and sunny days without precipitation as close as possible to each year of each stage of the BBCH.
• Yes, multi-annual evaluation is an average of 100 years, we have added this information.
• We evaluated the results according the developmental stage using BBCH-scale instead of seasonality
• L165: When the leaves were fully expanded in spring, the stomatal conductance and transpiration rate decreased significantly, but the photosynthetic rate tended to increase till summer (begging of apple maturity).
• In line 168 we specified by BBCH scale.

We supplemented the data on the age and size of apples in the methods, as well as clarified the measurement methods.

The effect of low night temperature on photosynthesis intensity has not been studied, but it could be task for future research. Thank you very much for your remarks.

Thank you very much for all your comments and suggestions for improving the quality of our manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript has improved reasonably from the previous version. 

The authors' responses to reviewer comments could be better organised and should address ALL the comments rised by the reviewer.

The paper now is suitable for publication.

Author Response

Dear reviewer,

Thank you very much for all the comments to improve the quality of the manuscript and to enrich the experience of the authors.

I am very sorry if we did not respond to all your comments...

Respectfully authors

Author Response File: Author Response.pdf

Reviewer 2 Report

After carefully reviewing the manuscript I concluded that the authors sufficiently addressed my comments.

Therefore I suggest that the article is accepted for publication in Agronomy.

Thank you for giving me the opportunity to assess the manuscript.

Author Response

Dear reviewer,

Thank you very much for all the comments to improve the quality of the manuscript and to enrich the experience of the authors. Young researchers have the opportunity to improve and learn how to write high-level articles only because of such a thorough review as yours.

Respectfully authors

Author Response File: Author Response.pdf