Effects of Water Deficit Stress on Growth Parameters of Robinia pseudoacacia L. Selected Clones under In Vitro Conditions

Round 1

Reviewer 1 Report

Manuscript Number: forests-2049724-peer-review-v1

In this manuscript entitled “Effects of water deficit stress on growth parameters of Robinia pseudoacacia L. selected clones under in vitro conditions” the authors used a plant tissue culture technique to assess the phenotypic response of three highly productive genotypes of Robinia pseudoacacia to water deficit induced by mannitol and sucrose in a range of water potentials from 0 MPa to -1.5 MPa in an eight-week experiment. Subsequently, the authors through systematic analysis of growth parameters by two models, it is proved that genotype-specific responses to induced drought stress, indicating the potential for tree improvement in productivity and stress tolerance. All in all, it is a good worked-out manuscript, but in my opinion, some shortcomings should be resolved.

Materials and Methods

Line 137-139: “Three genotypes of R. pseudoacacia characterized by good stem form, high productivity and vigour” What are the criteria for selecting the good stem form, high productivity and vigour? How many genotypes of R. pseudoacacia in the forest stands? Please clearly described.

Line 222: changed “SAS/STAT® v. 14.3” to “SAS/STAT v. 14.3”

Results

Line 232: Table 3: changed “p-value” to “p” in the first row of table.

Line 300: Table 7: changed “p-value” to “p” in the first row of table.

References

There are to many problems in this part, please check all references carefully. eg: Latin requires italics, problem of format and so on. Check one by one and modify it.

Line 597: Science (80-. ) ?

Line 637-639: changed “Robinia pseudoacacia L.” to “Robinia pseudoacacia L.”

Line 640-642: changed “Robinia pseudoacacia L.” to “Robinia pseudoacacia L.”

Line 643-645: changed “Robinia pseudoacacia L.” to “Robinia pseudoacacia L.”

Line 648-649: Please added the number of doi.

Line 650-652: changed “Robinia pseudoacacia L.” to “Robinia pseudoacacia L.”

Line 667: changed “Lawlor DW” to “Lawlor, D.W.”

Line 701-702: changed “Liang, K.-Y.” to “Liang, K.Y.”

Author Response

Review 1

Dear Reviewer,

Thank you for your comments and suggestions to improve our manuscript. All of your suggestions have been considered and all of them were incorporated into the manuscript. Please read the responses to your comments below.

 

1. Line 137-139: “Three genotypes of R. pseudoacacia characterized by good stem form, high productivity and vigour” What are the criteria for selecting the good stem form, high productivity and vigour? How many genotypes of R. pseudoacacia in the forest stands? Please clearly described.

 

All three genotypes of R. pseudoacacia were selected by the National Commission as plus trees phenotyped according to the National Standards for the Selection of Forest Trees. According to the standards, these trees are characterised by desirable traits such as stem form (straightness), high productivity (well above the average for the region), and vigour. Plus trees are selected only in selected seed stands or at least in production seed stands of selected species where the species is dominant.

2. Line 222: changed “SAS/STAT® v. 14.3” to “SAS/STAT v. 14.3”
3. Line 232: Table 3: changed “p-value” to “p” in the first row of table.
4. Line 300: Table 7: changed “p-value” to “p” in the first row of table.
5. There are to many problems in this part, please check all references carefully. eg: Latin requires italics, problem of format and so on. Check one by one and modify it.
6. Line 597: Science (80-. ) ?

 

It was corrected: Zhao, M.; Running, S.W. Drought-Induced Reduction in Global Terrestrial Net Primary Production from 2000 Through 2009. Science. 2010, 329, 940–943, doi:10.1126/science.1192666.

 

7. Line 637-639: changed “Robinia pseudoacacia L.” to “Robinia pseudoacacia L.”
8. Line 640-642: changed “Robinia pseudoacacia L.” to “Robinia pseudoacacia L.”
9. Line 643-645: changed “Robinia pseudoacacia L.” to “Robinia pseudoacacia L.”
10. Line 648-649: Please added the number of doi.

It was corrected: Polle, A.; Chen, S.L.; Eckert, C.; Harfouche, A. Engineering drought resistance in forest trees. Front. Plant Sci. 2019, 9, doi: 10.3389/fpls.2018.01875

11. Line 650-652: changed “Robinia pseudoacacia L.” to “Robinia pseudoacacia L.”
12. Line 667: changed “Lawlor DW” to “Lawlor, D.W.”
13. Line 701-702: changed “Liang, K.-Y.” to “Liang, K.Y.”

 

All of your minor suggestions (2-5, 7-9 and 11-13) have been also corrected in manuscript

Author Response File: Author Response.docx

Reviewer 2 Report

General recommendations

1.      Provide graphical abstract for the paper which represents the whole scheme of study.

2.      Why you have selected Robinia pseudoacacia L.? Why not any other economically important plant?

3.      Why you have selected three genotypes not more?

Abstract

1.      It should be start with a brief background, then aims of the study, methodologies, results and conclusion.

Introduction

1.      Please clearly write your objectives at the end of the introduction.

Methods

Plant material and experimental design

1.      The cultures were maintained for eight weeks to investigate their growth potential and response to drought stress. Why not more time?

RESULTS

Effect of water stress on mortality

1.       All three R. pseudoacacia genotypes studied showed different behaviour in terms of mortality rate depending on the duration of drought stress, osmoticum type and its concentration. What is the reason?

2.       The highest mortality rate was observed for the clone 4SO especially for the osmoticum below -0.6 MPa. What is the reason for selecting this method?

3.       The highest mannitol concentration (120 gl-1 241 ) caused a plant mortality rate of 20% in 6SO and 12% in 10PT, which was also sensitive to the highest concentration of sucrose (S90).. What is the reason?

4.       For treatment S60, we observed virtually no plant mortality, regardless of genotype or treatment duration. Revise this sentence.

Effect of drought stress on total shoot length

1.       However, the largest relative TSL increment 305 under stress conditions was observed with the 6SO clone. What is the reason?

2.       In turn, sucrose S90 in the 10PT clone, did not cause such an inhibitory effect as observed in the 6SO clone. What is the reason?

Effect of drought stress on total fresh weight

1.       The most significant increase in FW was recorded for sucrose osmoticum, in particular, concentration of 30 gl-1 produced the highest results of FW, which was particularly evident for 4SO and 6SO clones. What is the possible reason?

Conclusion

1.      Please rewrite the conclusion which includes highlights of your results.

2.      What do you recommend for the future?

 

3.      What are the benefits of this study?

Author Response

Review 2

Dear Reviewer,

Thank you for your comments and suggestions to improve our manuscript. All of your suggestions and questions about the manuscript have been considered and our responses to your specific questions can be found below. As you suggested, we prepared graphical abstract. We have incorporated some of the suggestions into the manuscript. We have clarified your other questions in our responses below. Please read the responses to your comments below. We strongly believe that our responses to your questions are sufficient and that you can accept our manuscript based on these clarifications.

 

1. Provide graphical abstract for the paper which represents the whole scheme of study.

 

As you suggested, in the revised manuscript we provide graphical abstract for the study.

 

2. Why you have selected Robinia pseudoacacia L.? Why not any other economically important plant?

We selected Robinia pseudoacacia because it is already an economically important species in some European countries (e.g., Hungary) and its importance is increasing in Central Europe, as the species is considered a "winner" in Central Europe under projected climate change scenarios (Dyderski et al. 2017). Moreover, we have already pointed out the economic importance of this species in the introduction, lines 77-85.

 

3. Why you have selected three genotypes not more?

Thank you for this question. This limitation on the number of clones is due to our study's aims and the methodology we chose. Above all, we selected only three clones of R. pseudoacacia to gain a better understanding of the behavioural patterns and to determine the appropriate methodology to support the future selection of drought-resistant clones on a larger scale. Therefore, in this study, we do not show the results of large-scale screening programmes, but focus on complete control of culture conditions and monitoring the influence of a single factor, which allow us to develop and made recommendations for breeding programmes.

 

Abstract

 

1. It should be start with a brief background, then aims of the study, methodologies, results and conclusion.

Word count limitation in the abstract does not allow us to elaborate on this part of the manuscript. We also believe that the current version of the abstract contains sufficient information regarding the background of the study, aims, methodology, results and conclusions.

Introduction

 

1. Please clearly write your objectives at the end of the introduction.

The objectives of our study are: to measure the variation in genotype response to a drought-induced event in order to gain a better understanding of behavioural patterns and to support the future selection of drought-resistant clones of R. pseudoacacia. We put this sentence at the end of the last paragraph of the introduction, as you suggested.

Methods

 

Plant material and experimental design

 

1. The cultures were maintained for eight weeks to investigate their growth potential and response to drought stress. Why not more time?

The length of the study allowed us to observe already significantly different responses of the clones to the treatments. Moreover, the end of the experiment was determined by the mortality of the tested clones of R. pseudoaccacia .

 

RESULTS

 

Effect of water stress on mortality

 

1. All three R. pseudoacacia genotypes studied showed different behaviour in terms of mortality rate depending on the duration of drought stress, osmoticum type and its concentration. What is the reason?

We broadly write about it in the paper as follows:

“Under the lowest water potential (-1.5 MPa), mortality was observed in all studied clones, although tolerance to stress varied widely among the three clones. In fact, clone 6SO showed little mortality until exposed to the most severe stress conditions (-1.5 MPa), whereas clone 4SO was far less tolerant to stress with mortality occurring at only -0.3 MPa.

McDowell et al. [53] showed that isohydric species are more likely to die during prolonged droughts of moderate intensity, while anisohydric trees such as R. pseudoacacia are more likely to die during intense, even short, droughts. In our study, R. pseudoacacia clones showed considerable variation in their response to drought and its duration. The 6SO clone was characterized by maintaining the highest survival rate among the clones tested throughout the experimental period and the occurrence of mortality was noted only under the most severe drought stress conditions beginning in the fourth week, while the 4SO suffered drought-induced death as early as the second week under mild to moderate drought. Although we did not consider stomatal conductance and xylem cavitation in our study, the varied mortality and growth responses to drought of the clones studied in our experiment support the hypothesis that genotypes rather than species should be considered with respect to specific physiological strategies.”

 

2. The highest mortality rate was observed for the clone 4SO, especially for the osmoticum below -0.6 MPa. What is the reason for selecting this method?

The issue you raised broadly discusses it as follows “Water deficit induced by mannitol or sucrose is analogous to drought conditions in the natural environment [38] and can be characterized by a decrease in soil water potential (Ψ). Values of Ψ from 0 to -0.3 MPa are typical for well-watered plants, whereas values below -0.4 MPa corresponds to moderate water stress, and values from -1.5 to -2.0 MPa represent severe stress and permanent loss of turgor in most plant species. Water potentials below -2.0 MPa are likely to cause severe vascular embolism. It should be noted, however, that these values vary depending on the species and drought model [42] and not only Ψ-decrease affects the plant, but also its duration affects the extent of the damage. Many studies have attempted to determine the threshold water potential that suppresses plant growth [55–57]. For woody plants, a soil water potential threshold below -0.3 MPa can be expected to inhibit plant growth [57]. Ridolfi and Dreyer  (1997) found a drought threshold of -0.6 MPa for Populus × canadensis ‘Robusta’. This negative plant response threshold can be used as a guide for selecting tolerant genotypes, below which fast-growing trees lose their advantage. Our study showed that intense drought indeed triggered a rapid inhibition of shoot length growth, while under mild stress (-0.2 MPa) all clones studied achieved greater TSL versus the control treatment, indicating an increase in water use efficiency under mild stress or an effect of the specific osmotic agent (sucrose) discussed below. Importantly, the responses of the three black locust clones studied were nuanced and depended on the clone (genotype), the type of osmotic agent, and its concentration, combined with the exposure time under stress conditions. The 4SO clone was the most drought sensitive among the clones studied. It was characterized by the lowest TSL increment and the highest mortality rate under moderate stress conditions (80% at week eight at a water potential of -0.6 MPa). The 10PT clone was characterized by an intermediate response to stress-induced conditions. Clone 10PT showed stable growth at a water potential of -0.6 MPa, however, mortality reached 57% after eight weeks under moderate stress conditions (at a water potential of -0.6 MPa). The highest drought resistance among the clones studied was shown by clone 6SO, which not only achieved the greatest TSL during eight weeks of drought but also had the lowest mortality (3% at a water potential of -0.6 MPa).

 

3. The highest mannitol concentration (120 gl-1 241 ) caused a plant mortality rate of 20% in 6SO and 12% in 10PT, which was also sensitive to the highest concentration of sucrose (S90). What is the reason?

We broadly discuss this issue as follows: “The three clones responded differently to the osmoticum and its concentration. In general, mannitol had a stronger inhibitory effect on TSL and FW increment on the black locust clones compared with sucrose. Our experiment showed that TSL and FW increment was suppressed immediately after exposure to mannitol, regardless of its concentration in the medium (120, 90 and 25 gl-1). In the case of mannitol exposure, our results are consistent with the findings of Claeys et al. [36], who indicated that plant growth is extremely sensitive to mannitol and that growth rates decreased rapidly when plants are exposed to even low concentrations.

In the present study, mannitol not only suppressed plant growth but was also responsible for the sharp increase in mortality of R. pseudoacacia clones, confirming that sucrose and mannitol play different roles in plant metabolism. Unlike mannitol, sucrose can be taken up and metabolized by the plant. At low concentrations (2% and 4%), sucrose is necessary for optimal growth and reproduction and can increase dry weights. The fact that we observed growth inhibition at low-stress levels with sucrose-containing media (S90), fits with the common view that stress-induced growth inhibition is an active process that is not dependent on carbon restriction, as was the case in our experimental conditions, where sucrose acted as a carbon source in the plant medium, significantly promoting the growth of clones in in vitro culture at a concentration of 30 gl-1 (S30). However, further increasing sucrose supply reduced the water potential in the leaves in a dose-dependent manner in the leaf tissue. A concentration of 90 gl-1 (S90 ~-0.6 MPa) caused a negative effect, as evidenced by significantly lower TSL and FW growth compared to the S30 treatment, while significantly increasing tree mortality. The results obtained are consistent with previous findings of Hoekstra et al. [65], who showed that prolonged heat and drought stress leads to a progressive accumulation of sucrose, a high concentration of which reduced the growth rate in the plants. In this context, our results showed different metabolic pathways of sucrose, depending on the concentration of sucrose in the medium, and generally different responses to the osmoticum treatments and the overall concentrations among the three clones tested, with clone 6SO being the most tolerant to drought stress, regardless of osmoticum or concentration.”

 

4. For treatment S60, we observed virtually no plant mortality, regardless of genotype or treatment duration. Revise this sentence.

Thank you for this comment, in the revised manuscript it was corrected as follows: Plant mortality did not occur with the application of S60, regardless of genotype and treatment duration (Table 6).

Effect of drought stress on total shoot length

 

1. However, the largest relative TSL increment 305 under stress conditions was observed with the 6SO clone. What is the reason?

We refer to this result as follows: “This clone maintained its growth even under the lowest water potentials in the medium, increasing its TSL by 13% compared with the initial TSL in M120 and M90 media, while growth was most inhibited in S90, increasing by only 8% of the initial TSL. This suggests that clone 6SO had the highest drought resistance among the clones tested, not only achieving the highest TSL during the eight weeks of drought, but also the lowest mortality (3% at a water potential of -0.6 MPa).”

2. In turn, sucrose S90 in the 10PT clone did not cause such an inhibitory effect as observed in the 6SO clone. What is the reason?

Sucrose and mannitol play different roles in plant metabolism. Unlike mannitol, sucrose can be taken up and metabolised by the plant. In this context, our results showed different metabolic pathways for sucrose also depending on the genotype. We write about this in lines: ...

Effect of drought stress on total fresh weight

 

1. The most significant increase in FW was recorded for sucrose osmoticum, in particular, a concentration of 30 gl-1 produced the highest results of FW, which was particularly evident for 4SO and 6SO clones. What is the possible reason?

Sucrose can be taken up and metabolized by the plant. At low concentrations (2% and 4%), sucrose is necessary for optimal growth and reproduction, so our scale for sucrose starts at this concentration optimal for the growth of R. pseudoaccacia clones. - which was already stated in lines ….

Conclusion

 

1. Please rewrite the conclusion which includes highlights of your results.

We are not sure which part of the conclusion section you have in mind.

2. What do you recommend for the future?

As we highlight in the conclusions, our study revealed genotype-specific responses to induced drought stress. This suggests the potential for tree improvement programs to select on both productivity and stress tolerance, which is particularly important given climate change with more frequent drought events. In addition, for large-scale breeding programs designed to select drought-tolerant genotypes, we recommend the use of culture media containing 90 gl-1 mannitol or 90 gl-1 sucrose at an early selection stage to obtain early screening results. In the conclusions, we also suggest that the potential applicability of the in vitro rapid screening method in tree improvement programs should be further confirmed by testing under field conditions, preferably at multiple test sites, to evaluate genotype-by-environment interactions and assess the long-term impact of utilizing this early selection strategy. See lines 527-548.

3. What are the benefits of this study?

The most important and immediate benefit of our in vitro experiment, in which we used a wide range of water potentials, is that this experiment allowed us to rapidly screen three R. pseudoacacia clones under mild to life-threatening drought stress conditions. The other advantages of our study demonstrate the potential of this method for large-scale breeding programs aimed at selecting drought-tolerant genotypes at an early growth stage (early selection strate

Author Response File: Author Response.docx