Abscisic Acid Priming Creates Alkaline Tolerance in Alfalfa Seedlings (Medicago sativa L.)

Round 1

Reviewer 1 Report

Alkaline soils cause severe wilting and oxidative damages in plants, and disrupt ion balance and osmotic homeostasis. In the present study, Wei et al. showed that ABA pretreatment significantly enhances tolerance to alkaline stress in alfalfa seedlings by decreasing ROS accumulation, increasing the antioxidant enzymes activity, changing ion and osmotic homeostasis, and up-regulating the expression of stress tolerance-related genes.

The topic of the manuscript is suitable for Agriculture journal. English writing is good, material and methods are accurately described, and the results could be interesting for the scientific community.

In my opinion, the manuscript worths to be considered in Agriculture journal after the revisions listed below.

- Paragraph 3.4: Authors should clearly explain why only the expression levels of APV and NHX1 genes were reported, while results for POD, CuZnSOD and P5CS genes were not shown. Even if the expression results for these three genes are not interesting for the paper, they should be reported at least in the Supplementary material.

- Species scientific names (i.e. Medicago sativa) and genes names (i.e. APV, NHX1, etc.) should be written in italic font.

- Line 162: the authors should clearly explain why did they choose Actin2 as housekeeping gene.

- Line 16: insert a comma between the words “stress” and “and”.

- Line 18: change “simulated by” with “using a”

- Line 65: add a space after the full stop

- Line 124: “plant” should be plural (plants)

- Line 159: “gene” should be plural (genes)

- Line 214: please change “more” with “higher”

- Line 307: please explain in detail the “range of stresses” against which priming treatment increase tolerance.

- Line 318: please delete the reference “Cao et al. 2013” (it should be the number [45])

In general, an accurate English revision is required in order to avoid minor errors and further improve the manuscript readability.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

In this paper the authors used abscisic acid (ABA) pretreatment as a priming agent to increase the tolerance of alfalfa seedlings to alkaline (Na₂CO₃) stress. It was shown that ABA pretreatment decreased the reactive oxygen species (ROS) accumulation, increased the proline content and the activitiy and gene expression of some antioxidative enzymes, maintained higher cation’s ratios in alfalfa plants under the used stress treaments. The manuscript contains valuable information, but there are several problems with it in the present format.

Questions and critical remarks:

• The manuscript deals with the alkaline tolerance of plants, thus it is necessary to give more information about the pH in different solutions and its changes in function of time, and about its adjustment. What was the pH in the control and ABA-containing solutions? How did the pH change during the treatments?
• The description of the used methods sometimes is not correct or not clear (e.g., lines 94-95). There is a detailed information about the preliminary experiments to choose the right Na₂CO₃ and ABA concentrations, but the results are shown only in the Supplementary Material. I suggest shortening of this section (lines 101-111) and relocate the relevant information into the Supplementary Material.
• The used abbreviation of treatments in the text and in the legends of figures are unusual. Why did you used CK for control (unstressed) plants?
• Because the “priming” originally was used in the case of biotic stresses, I suggest to mention some other phrases for it (such as hardening, sensitization) at least once.
• The manuscript contains several formatting, typing and linguistic mistakes. Its English should be improved. Some examples are:

line 52 (format), line 72 (grammar), lines 132-136 (format), line 159, lines 181-183 (grammar), line 189 (format), lines 220, 233, 250 (format), lines 246-248 (grammar), line 261 (format; the name of genes is written in italics; check through the manuscript), 288 (grammar), line 300 (typing), line 315 (grammar), lines 324-325 (rephrase the sentence), 343 (grammar)

Author Response

Please see the attachment

Author Response File: Author Response.pdf