Water Deficit Severity during the Preceding Year Determines Plant Tolerance to Subsequent Year Drought Stress Challenges: A Case Study in Damask Rose

Round 1

Reviewer 1 Report

The authors used damask rose as materials and conducted the field experiments under three water deficit levels. The plant phenology and physiological indexes were monitored across two years. The results in this manuscript provided a basic understanding of the rose plants in response to drought stress. Some comments are listed below and need to be addressed. 

The text field condition of the experiment 

1. More phenotypes of damask rose under subsequent year drought stress should be provided to gain more insight evidence.  Because the authors concluded that the decrease in leaf area was mediated by reductions in both the number of leaves and individual leaf area. How did the author count the number and measure the leaf area？The graphic figure of the representative indexes could better diminish the misunderstanding.

2. The soil characteristics for the field study should be provided in Section 2.1.

3. How did the author calculate the three water deficit levels (70, 40, and 10% available water content)? How did the author keep water deficit levels consistent for the damask rose plants in the field conditions? More phenotypes of damask rose plants (such as leaves, stems, flowers, etc.) should be provided to better display the experimental conditions. 

4. line 354-355, the author concluded that shoot dry weight was associated with the number of petals and receptacle diameter. What is the meaning of this finding? In Table 4, the petal dry weight (% of the total) exhibited lower content than that of the flower, which seems not unreasonable. What is the possible reason for this? In addition, the correlation indexes of each parameter should be included in the manuscript.

5. In Sections 3.2, 3.3, 3.4, and 3.5, the description of the results was too simple, and can not provide enough useful information for damask rose under different water-deficit levels.

6. The measured data or parameters (Table 1 to Table 6) presented in the manuscript should be means plus/minus standard error.

7. The section of the conclusion should summarize the main findings and objective of the manuscript, not list the main results. The authors should revise it.

English expressions should be improved. Authors should clearly distinguish the words 'drought' and 'water deficit' in the manuscript.

Author Response

Reviewer 1

The authors used damask rose as materials and conducted the field experiments under three water deficit levels. The plant phenology and physiological indexes were monitored across two years. The results in this manuscript provided a basic understanding of the rose plants in response to drought stress. Some comments are listed below and need to be addressed.

The text field condition of the experiment

Authors: The experimental layout is now described in detail (Lines 146-152), and presented as Figure (newly added Figure S3).

1. More phenotypes of damask rose under subsequent year drought stress should be provided to gain more insight evidence. Because the authors concluded that the decrease in leaf area was mediated by reductions in both the number of leaves and individual leaf area. How did the author count the number and measure the leaf area? The graphic figure of the representative indexes could better diminish the misunderstanding.

Authors: (1) Nine treatments [3 water deficit regimes (year 1) × 3 water deficit regimes (year 2)] were realized, and evaluations spaned for 3 successive years (2017−2020); (2) The associations among parameters under study is provided in Fig. 1; and (3) Details on the individual and whole-plant leaf area determinations are now provided (Lines 223-226, 229-230).

2. The soil characteristics for the field study should be provided in Section 2.1.

Authors: Soil characteristics are now provided (Lines 97-101).

3. How did the author calculate the three water deficit levels (70, 40, and 10% available water content)? How did the author keep water deficit levels consistent for the damask rose plants in the field conditions? More phenotypes of damask rose plants (such as leaves, stems, flowers, etc.) should be provided to better display the experimental conditions.

Authors: (1) The process of irrigation adjustment to different available water contents is now provided (Lines 137-145 and 153-157); and (2) Aboveground plant organs were separately assessed (leaves, stems, and flowers). Results are provided in Tables 3 and 4, and associations among them in Fig. 1.  

4. line 354−355, the author concluded that shoot dry weight was associated with the number of petals and receptacle diameter. What is the meaning of this finding? In Table 4, the petal dry weight (% of the total) exhibited lower content than that of the flower, which seems not unreasonable. What is the possible reason for this? In addition, the correlation indexes of each parameter should be included in the manuscript.

Authors: (1) Larger plants (higher shoot dry weight) bear bigger flowers. This increase in flower size is associated with larger receptacle (axis to which floral organs are attached) diameter, and higher number of petals (from which essential oil is extracted); (2) It is now clarified that this parameter is Dry weight ratio [(dry weight/fresh weight) × 100; provided in percentage], and not dry mass partitioning to the respective organ (Table 4); and (3) The associations among parameters under study is provided in Fig. 1.

5. In Sections 3.2, 3.3, 3.4, and 3.5, the description of the results was too simple, and can not provide enough useful information for damask rose under different water-deficit levels.

Authors: As indicated, the results were enriched accordingly (Lines 416-426, 442-444, 467-470, 474-475, 487-488, 506-508, 540-553).

6. The measured data or parameters (Table 1 to Table 6) presented in the manuscript should be means plus/minus standard error.

Authors: As indicated, standard error values were added in all mean values (Tables 1−6).

7. The section of the conclusion should summarize the main findings and objective of the manuscript, not list the main results. The authors should revise it.

Authors: As indicated, the conclusion section was revised and enriched (Lines 677-682).

Comments on the Quality of English Language

English expressions should be improved. Authors should clearly distinguish the words 'drought' and 'water deficit' in the manuscript.

Authors: The term ‘drought’ was replaced with the term ‘water deficit’ throughout the manuscript.

Reviewer 2 Report

The study certainly required a lot of effort; there are a lot of data and this is the major strength of the paper.

However, in my opinion, there is little clarity in the experimental design.

The experiment was conducted using cuttings rooted in vitro and subsequently transplanted into the open field. The different stress levels and related irrigation treatments are not based on the potential evapotranspiration of the crop, but on values taken from the literature, which, however, come from pot experiments. It is unclear how these values were adjusted for field conditions, and it appears that a pre-experiment was also conducted, but its context is unclear. Further clarification in this regard is needed.

The lack of measurements relating to plant and soil water potentials (md and pd potentials) makes it difficult to calculate and monitor the plants stress level, especially in an uncontrolled environment such as the open field. Although it was stated that the water level in the soil was monitored via probes, but it is not clear what level of stress for the plants this corresponds to.

Furthermore, for the various measurements, it is unclear the number of both biological (number of plants) and “analytical” replicates used. So, when exactly were the physiological measurements taken? Hours of the day, exact days...and on how many leaves per plant?

For biochemical measurements, how were the sampling done? What type of leaves were used? How many replications?

If it is written, it is unclear and must be specified clearly for each parameter.

Another thing that really needs some clarifications: in the paragraph relating to gas exchange in the materials and methods, it is stated that the PPFD used is 200 μmol m−2 s−1. This value is really too low to represent open field conditions. Even if it is stated that the measurements were taken in full sun, with this PPFD the leaves practically adapt to the shade. Was it 2000 and is it just a typo?

Furthermore, in same paragraph, the "mesophyll conductance" is referred to as the Pn/Ci ratio, which is incorrect. The conductance of the mesophyll is something completely different, and it certainly isn't measured like this, but respiration curves etc. are needed.

Even for the statistical analysis, the Anova applied is not clear to me, or perhaps I just misunderstood it.

Author Response

Reviewer 2

The study certainly required a lot of effort; there are a lot of data and this is the major strength of the paper.

However, in my opinion, there is little clarity in the experimental design.

The experiment was conducted using cuttings rooted in vitro and subsequently transplanted into the open field. The different stress levels and related irrigation treatments are not based on the potential evapotranspiration of the crop, but on values taken from the literature, which, however, come from pot experiments. It is unclear how these values were adjusted for field conditions, and it appears that a pre-experiment was also conducted, but its context is unclear. Further clarification in this regard is needed.

Authors: (1) The experimental layout is now described in detail (Lines 146-152), and presented as Figure (newly added Figure S3); (2) The process of irrigation adjustment to different available water contents is now provided (Lines 137-145 and 153-157); (3) Details on the preliminary study were now added (Lines 131-136).

The lack of measurements relating to plant and soil water potentials (md and pd potentials) makes it difficult to calculate and monitor the plants stress level, especially in an uncontrolled environment such as the open field. Although it was stated that the water level in the soil was monitored via probes, but it is not clear what level of stress for the plants this corresponds to.

Authors: The process of irrigation adjustment to different available water contents is now provided (Lines 137-145 and 153-157).

Furthermore, for the various measurements, it is unclear the number of both biological (number of plants) and “analytical” replicates used. So, when exactly were the physiological measurements taken? Hours of the day, exact days...and on how many leaves per plant?

Authors: The experimental layout is now described in detail (Lines 146-152), and presented as Figure (newly added Figure S3). Details on the number of replicates, period of sampling, and time of the day are now provided (Lines 146-152, 158-162, 167-172, 186-193, 201-207,  247-250, 263-264, 274, 278-279, 284, 296-297, 310-311, 325, 349).

For biochemical measurements, how were the sampling done? What type of leaves were used? How many replications?

If it is written, it is unclear and must be specified clearly for each parameter.

Authors: The experimental layout is now described in detail (Lines 146-152), and presented as Figure (newly added Figure S3). Details on the number of replicates, sampling procedure, and sampling tissue are now provided (Lines 146-152, 158-162, 167-172, 186-193, 201-207,  247-250, 263-264, 274, 278-279, 284, 296-297, 310-311, 325, 349).

Another thing that really needs some clarifications: in the paragraph relating to gas exchange in the materials and methods, it is stated that the PPFD used is 200 μmol m⁻² s⁻¹. This value is really too low to represent open field conditions. Even if it is stated that the measurements were taken in full sun, with this PPFD the leaves practically adapt to the shade. Was it 2000 and is it just a typo?

Authors: Indeed, it was a mistake, which was now corrected (Line 199).

Furthermore, in same paragraph, the "mesophyll conductance" is referred to as the P_n/C_i ratio, which is incorrect. The conductance of the mesophyll is something completely different, and it certainly isn't measured like this, but respiration curves etc. are needed.

Authors: As indicated, this term was erased (Line 204).

Even for the statistical analysis, the Anova applied is not clear to me, or perhaps I just misunderstood it.

Authors: The text was re-written (Lines 352-354).

Reviewer 3 Report

Line 18: In this field study, plants were subjected.... revise the statment such as "In the present study, plants were subjected.....

Add prominent results in the abstract section from all activities

The introduction is well-written; however, add some recent references.

I would also suggest adding relevant references in the discussion section.

I have a little concern about the Fv/Fm calculation. Kindly add some detail.

Chlorophyll fluorescence features of Rosa damascena ‘Kashan 93’ cultivated under different watering regimes (70, 40 and 10% available water content) during two consecutive years. elaborate Fv/Fm vs Fv/F0 in your results section.

Author Response

Reviewer 3

Line 18: In this field study, plants were subjected.... revise the statment such as "In the present study, plants were subjected.....

Authors: Done (Line 18).

Add prominent results in the abstract section from all activities

Authors: Done (Lines 22-27).

The introduction is well-written; however, add some recent references.

Authors: Done (Lines 56, 58, 75, 747-753, 773-780).

I would also suggest adding relevant references in the discussion section.

Authors: Done (Lines 610-616, 806-818).

I have a little concern about the F_v/F_m calculation. Kindly add some detail.

Authors: Done (Lines 178-180, 186-188, 413-417).

Chlorophyll fluorescence features of Rosa damascena ‘Kashan 93’ cultivated under different watering regimes (70, 40 and 10% available water content) during two consecutive years. elaborate F_v/F_m vs F_v/F₀ in your results section.

Authors: Done (Lines 413-424).

Round 2

Reviewer 1 Report

In the revised manuscript, the author added some needed descriptions and provided some basic information of the materials used for the field experiment, and addressed the concerns that I emphasized. While I still recommended the author to add some morphological of the rose plants in the main text Figures. These can benefit our readers and gain more interest in the same subject.

English is good, and some descriptions of the main results can be simple and concise.

Author Response

Reviewer 1

In the revised manuscript, the author added some needed descriptions and provided some basic information of the materials used for the field experiment, and addressed the concerns that I emphasized. While I still recommended the author to add some morphological of the rose plants in the main text Figures. These can benefit our readers and gain more interest in the same subject.

Authors: A new figure was added (newly added Fig. 2).

Reviewer 2 Report

The manuscript has been significantly improved by the authors' thorough responses addressing all raised concerns and is now ready for publication

Author Response

Reviewer 2

The manuscript has been significantly improved by the authors' thorough responses addressing all raised concerns and is now ready for publication.

Authors: Thank you.