Exogenous Melatonin Alleviates the Inhibitory Effect of NaHCO3 on Tomato Growth by Regulating the Root pH Value and Promoting Plant Photosynthesis
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsMelatonin, a potent antioxidant and plant regulator alleviates the damage caused by various stresses. The manuscript by Y.Yang and colleagues submitted for publication in “Agronomy” describes the effects of exogenous melatonin on tomato subjected to alkaline stress. Melatonin ameliorated reductions in plant growth and photosynthesis of tomato seedlings inhibited by increased pH in the root environment under stress conditions. The authors thoroughly studied the effects of exogenous MT application on the pH of the root environment as well as the parameters of photosynthetic gas exchange and chlorophyll fluorescence. While discussing the possible mechanisms of the positive effect of melatonin, the authors conclude that MT treatment led to the improvement of stomatal limitation of photosynthetic rate under NaHCO3 stress and increased the photosynthetic сapacity of tomato seedlings. Exogenous MT facilitated the electron transfer on the receptor side of PSII and improved alkali tolerance.
Despite a balanced discussion of the reasons for the positive effect of melatonin on photosynthetic parameters, the authors did not discuss possible mechanisms that could induce plants to secrete large amounts of acid to alter the pH of root environment under MT treatment. For example, in maize seedlings MT was shown to increase the content of potassium ions and decrease the content of sodium ions, thus maintaining the stability of ions under salt stress (Jiang et al. 2016). In addition, it remains completely unclear for readers, how “Exogenous melatonin mediates endogenous melatonin signaling” (4.1) since the manuscript does not contain any mention of the expression of putative MT signaling genes such as Cand2 or genes for the mitogen-activated protein kinase (MAPK) cascade (Wei et al., 2018, Lee and Back, 2018).
Minor issues:
Lines 67-68: “rol e” to” role”
Line 90: with nearly uniform …? and uniform growth were selected (missing a word)
Line “suing” to “using”
Lines 156-157 “p-arameters” to “parameters”
Line 318: “and even death in severe cases” to “and even causing death in severe cases”
Comments on the Quality of English LanguageMinor editing is reguired
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
I read your manuscript entitled "Exogenous melatonin application alleviates the inhibition of tomato growth by NaHCO3 via regulating the pH of root environment and promoting plant photosynthesis". It is a solid text about the relatively new plant regulator, melatonin, and it offers new insights into the effects of melatonin on plant growth under alkaline conditions.
Line 49 - Can the authors provide some information on the possible role of melatonin as a phytohormone, and how it might influence their findings?
Line 85 - what is "pure water"? Sterile double distilled water? Sterile tap water? Also "warm water" - same comment. Please, specify what you mean.
Line 87 - Please, provide additional info on light conditions in the greenhouse. How many hours of direct sunlight and how many hours of darkness were the plants exposed to? Was the illumination artificial at any point? If yes, specify these light conditions as well.
Line 93 - 95 Please, re-phrase these sentences. Were the plants transferred from 1/2 H medium to full strength (undiluted) H medium? Please, specify. Also, although the Hoagland medium is a conventionally and widely used medium, it should be referenced.
Line 101 Which tool did you use to apply the melatonin solution? Was it some form of plant mister, spray bottle, or sprinkler? This entire sentence must be re-written, as it is insufficiently unclear in its current form: "and the standard of spraying was that water droplets were just formed and dripped down". What does "just formed" mean? Please, re-phrase to make it clearer for the reader.
Line 122 Was MT determined from the plant extract? Please, briefly describe the extraction i.e. the plant material preparation method for your HPLC analysis. Also, how did you ensure that the foliar MT that you exogenously applied didn't interfere with your quantification of endogenous MT?
Line 128 How was the calibration curve construed? What is the range of concentrations you used for your calibration curve?
Line 300 (Figure 6) I think this figure should be more prominent and in better resolution, if possible.
Line 314 Please, rephrase this subtitle "Exogenous melatonin mediates endogenous melatonin signaling to alleviate the inhibitory effect of NaHCO3 stress on tomato seedling growth". You did not analyze signaling pathways in this study: therefore, this subtitle is too strong. However, you may use an appropriate reference in the text and offer possible explanations for MT action on different signaling pathways.
Line 324 Does this mean that the plants actively uptake the MT? Please, briefly discuss.
Line 334 What are possible mechanisms by which MT ameliorates pH in the proximity of the roots? This must be included in the discussion. In my opinion, there is no need to reference Fig. 2 at this point, as it is already in the results.
Line 338 It is advisable to connect the discussion with the introduction more closely. In this case, you should use the same reference you used in the Introduction, to support your claims.
Line 348 Can you provide possible explanations for such effects of MT on chlorophyll content (and carotenoids)?
Line 383 Again, can you provide possible explanations for such effects of MT on photosynthesis? What is the possible mechanism? Is it due to the antioxidative properties of MT? Is it due to its direct binding to appropriate receptors?
At the end of the Discussion section, please add a short comment on the limitations of your study and what should be done in the future to expand your findings and strengthen your theory about the effects of MT on photosynthesis and tomato plant growth under alkalinized conditions.
References - please, check all the references and make sure that you truly need all of them. For the article of this scope, I believe more than 40 references are excessive.
Comments on the Quality of English LanguageEnglish language requires minor editing.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have considerably improved the manuscript by adding the results of the experiments, in which they measured the content of organic acids. This allowed them to put forward an ingenious hypothesis about the mechanisms of pH reduction in root environment. They also removed the part associated with melatonin signaling, limiting themselves to stating the mere fact that the level of expression of melatonin synthesis genes increased under the influence of exogenous melatonin.
However, I strongly recommend language editing by the native speaker, especially the newly inserted parts. I have noticed only some shortcomings, though grammatical errors may be throughout the manuscript.
In particular:
Lines 148-152 When describing methods, the indicative mood should be used, not the imperative. The same is true for the description of methods for determining the content of organic acids in the root medium (Lines 165-1870)
Lines 215 «Photosynthetic gas exchange parameters»” needs to be removed from the title of the chapter
Line 407 “ biosynthesises” should be changed for “ induces” or” upregulates”
Line 431 “indicating” to” indicates”
Comments on the Quality of English LanguageLanguage editing is required
Author Response
Please see the attachment
Author Response File: Author Response.pdf
