Exogenous 5-Aminolevulinic Acid Promotes Osmotic Stress Tolerance of Walnuts by Modulating Photosynthesis, Osmotic Adjustment and Antioxidant Systems

Round 1

Reviewer 1 Report

The MS entitled "Exogenous 5-aminolevulinic acid promotes osmotic stress tolerance of walnuts by modulating photosynthesis, osmotic adjustment and antioxidant systems " is a nice piece of work done by the authors.

Salinity affects plant processes and growth by imposing osmotic stress and destroys ionic and redox signalling. It also affects phytohormone homeostasis, which leads to oxidative stress and, eventually, imbalances in metabolic activity. In this situation, signalling compound crosstalk such as gasotransmitters [nitric oxide (NO), hydrogen sulfide (H2S), hydrogen peroxide (H2O2), calcium (Ca), reactive oxygen species (ROS)] and plant growth regulators (auxin, ethylene, abscisic acid, and salicylic acid) have a decisive role in regulating plant stress signalling and administer unfavourable circumstances including salinity stress.
The science structure and overall theme of the manuscript are sound and acceptable.

In my opinion, the overall concept is interesting and important. The paper is well written and is a worthy contribution that will be of interest to the readers of Forest Journal.

ALA application increased photosynthetic capacity and stimulated osmotic adjustment and antioxidant systems. All these were beneficial in improving walnut drought tolerance, but the mechanisms triggered by ALA should be further investigated in plants.

The paper requires only Minor revisions to be acceptable for publication, and I have a few minor suggestions which I believe will improve the manuscript.
The abstract can serve as a stand-alone document briefly describing procedures and conclusions. Highlights are specific and present new insights. In general, the paper is clear and well-written.

My suggestion regarding this MS is to accept after revising with the following points:
1. Authors presented a very good introduction, but they could explain more about plant osmotic tolerance's molecular mechanisms and potential dehydration mitigation.
2. Introduction and discussion sections authors could also involve new aspects - hormonal regulation of stress tolerance (interaction with SA, CYT etc.), including further references.
3. Add more view on photosynthetic parameters/discuss which kind of parameters are more sensitive and why.
4. Add some recent references in the MS. It is important to discuss the plant regulation mechanisms.
The paper brings many new aspects, and the novelty of the paper is OK, but I would like to invite authors to discuss more eco-physiological aspects using new references: DOI: 10.1016/j.plaphy.2022.08.024; 10.3389/fpls.2022.933694; 10.3389/fpls.2023.1129114; 10.1016/j.envres.2023.116292; 10.1007/s00344-021-10507-y; 10.1016/j.niox.2019.11.002

Final COMMENTS
- The manuscript is useful and innovative; it contains original data.
- This study presents the relevant matter in more depth than some related publications; therefore, I recommend its publication after MODERATE  changes.

Author Response

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Reviewer 2 Report

The submitted manuscript deals with the still current issue of the influence of water deficit on plants. The use of biologically active substances such as ALA is highly topical. As a model plant, the king walnut tree, juvenile stages of development, was chosen. Unfortunately, it is not entirely clear from the text which development phase this is. Only the size of the saplings is indicated. Furthermore, it is not entirely clear whether these are grafts or seedlings. In the case of grafted plants, the rootstock is also of considerable importance. This fact could also be mentioned in the introduction. Furthermore, it is necessary to realize that the water deficit, i.e. the drought induced by PEG, is not a water deficit, but an osmotic stress. Therefore, it would be appropriate to modify the title of the manuscript. The composition of Hoagland's solution is not stated in the methodology, as there are several variants and modifications of it. Furthermore, it is necessary to supplement the growing conditions (temperature, lighting, day length). Why were the trials established in the winter months? Weren't the plants dormant? The results are supported by graphs and tables. The results are clearly described, it is only a pity that correlation analyzes of individual characteristics were not created. The connection of osmotically active substances and RWC would be very interesting.

Author Response

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Reviewer 3 Report

   The manuscript by Zhong et al. was devoted to investigation of role of exogenous 5-aminolevulinic acid in the tolerance of walnuts to osmotic stress. It is interesting and complex work; however, I have several comments and questions.

   1. Introduction: If it is possible, mechanisms of action of 5-aminolevulinic acid should be described in more details.

   2. Line 80: Why was 10 mg L-1 ALA solution used? What was basis of using this concentration?

   3. Section 2.3: What were spectral characteristics of the actinic light used?

   4. Section 2.5: What was duration of the dark adaptation before the OJIP measurement?

   5. Line 118: Hansatech seems to be localized in UK.

   6. Figure 2: Some results seem to be contradictory. E.g., PEG+ALA significantly increased stomata conductance in comparison with action of PEG only. However, transpiration rates were similar in these variants. It is not clear. Additionally, leaf temperatures were similar in control and PEG+ALA variants; in contrast, this temperature was increased in the PEG variant. Authors explained these differences through differences in the stomata conductance. However, it is not probable because transpiration rate directly influence leaf temperature (changes in stomata conductance influences through changes in this rate), and the transpiration rate was not differed in the PAG and PAG+ALA variants.

   7. Section 3.3 (results of OJIP-test) shows that influence of PEG and PEG+ALA can be independent on changes in gas-exchange. It should be discussed in more detail.

Author Response

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