Physiological, Cellular, and Transcriptomic Analyses Provide Insights into the Tolerance Response of Arundo donax to Waterlogging Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Physiological, cellular and transcriptomic analyses provide insights into formation of adventitious roots in Arundo donax under waterlogging stress” provides information to improve the general knowledge about the waterlogging resilience response of this species.

 

MAJOR REVISION

Title: The title does not represent the obtained results. It should be more related to the whole plant resilience response.

Introduction: The objective of the study is not enunciated.

Materials and Methods: Please provide concise information regarding the experimental conditions. Include the number of plants per treatment, environmental conditions (soil temperature, aerial temperature, relative humidity, PPFD, photoperiod), pot size, substrate composition, etc.

Conclusions: In the conclusion section, the authors propose a model to explain the molecular mechanisms involved in the resilience response to waterlogging. In my opinion, there is not enough data to sustain this modelling. Their conclusions are only based on transcriptomic analysis and the relevant bibliography. More studies are necessary to clarify the variation in hormone signalling, metabolism, and enzyme activity in response to the strain.

English grammar: It is necessary to revise the English grammar in the whole manuscript thoroughly.

 

MINOR REVISION

Introduction

Although this section is informative, it is very long. Please consider shortening.

 Materials and Methods

Line 150: Please consider replacing “Experimental Materials and Treatments” with “Waterlogging stress treatment” and include the duration of the strain at the end of the paragraph (move here lines  168-169).

Lines 151-155: This paragraph is not clear. Please rewrite.

Lines 159-161: Does the stress treatment include an aeration system? . For example, agitation or forced ventilation?. Was the dissolved oxygen quantified from day 5^th to 45^th? This parameter is essential and probably determines the resilience responses.

Lines 163-165: This paragraph is not clear. Please rewrite.

Lines 166-172: Please rewrite.

Line 185: Please consider replacing “Paraffin section” with “Histological analysis” or something like that.

Lines 209-210: Please rephrase the paragraph “The roots (ARs) of 30d and 45d treatments and the control A. donax roots at 30 and 45 d were selected” to improve its understanding. Example: Total RNA were isolated from unstressed and stressed roots after 30 and 45 days of incubation. Samples were collected in triplicate …..  

 

Results

Lines 247-258: Please improve grammar. It is not easy to understand.

Lines 257-258: The new adventitious roots emerged from new sprout formation due to the imposed stress. Isn’t it?

Lines 347-473: Please avoid discussing the results in this section.

 

Discussion

Since the KEGG analysis revealed significant enrichment of pentose phosphate and tricarboxylic acid pathways (lines 305-308), it should be further discussed in this section.

Line 478: What is the meaning of “root rotting”?

Line 480: Please use the correct technical terminology. Replace “germination” with “sprouting”. The bud does not “germinate” …

Line 516: Consider replacing “… a reduction in the concentration of ABA” with “… a reduction in the free ABA concentration”.

Line 520: Consider replacing with free ABA content. The ABA excess is probably conjugated to other non-active forms or catabolized into phaseic acid.

 

Conclusions

 

Line 582: Consider replacing “fully watered plants” with “non-stressed plants”. 

Comments on the Quality of English Language

It is necessary to revise the English grammar in the whole manuscript thoroughly.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Congratulation

Just a couple comments; to be specific in the objective and conclusion, as remarked in the manuscript

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The submitted Manuscript describes the results of a well rounded study how the adventitious roots are formed in a herbaceous ornamental plant Arundo donax under waterlogging stress. The study involves cytology, morphology, biochemistry and transcriptomics. The results are worth to be published. The Reviewer likes the complex well-biased approach and the well-polished submitted Manuscript.

Some minor or major revision and amendments are needed before the publication. The Reviewer suggests major revisions for providing more time for corrections

 

1) Figure S1. Pls, provide the scale bar and units for the scale bar.

 

2) Table S4.

GO:0005372

water transmembrane transporter activity

0.000230587

 

What is it, a water channel or anything else?

 

3) Abstract.

 enzymatic ability of 22 the protective antioxidants (SOD, POD and CAT) in ARs also was greatly 23 enhanced as well after waterlogging. At the transcriptomic level, the 24 e

 

SOD, POD and CAT are enzymes but not antioxidants. Pls, correct.

 

4) Waterlogging events are becoming more often due to global 46 climate change [3,4].

 

More frequent, not more often.

 

5) Pls, read and cite the paper https://www.researchgate.net/publication/225790053_Plant_Anaerobic_Stress_as_a_Novel_Trend_in_Ecological_Physiology_Biochemistry_and_Molecular_Biology_1_Establishment_of_a_New_Scientific_Discipline or a simialr one from this Author.

 

6) 2.1. Experimental Materials and Treatments 150

A uniformly sized, well-established A. donax with axillary 151 buds was selected, and the culms were cut into 15 cm long 152 segments.

 

Pls, indicate where the plants were taken, location.

 

7) length of the 196 aerenchyma and area of the pericycle were measured by ImageJ

 

Please, indicate and describe how it was determined. In the results section, pls, describe in more detail the distribution of the aerenchyma spaces (uniform, uneven) and how the resulting parameters were measured.

 

8) Figure 5D. The scale bars and the units are not seen. Pls, change.

 

9) normally. The plants were maintained a ell-controlled glasshouse 163 with controlled light (16/8 h light/dark) and temperature (25/18C; 164 day/night) conditions.

 

a) Well-controlled, not ell-controlled.

b) Please, indicate the illumination intensity, humidity.

 

10) Pls, provide all the details for methods, states for the companies etc.

Comments on the Quality of English Language

Reasonable.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Despite the authors' diligent efforts to enhance the manuscript, the revised version still exhibits notable weaknesses that may hinder its acceptance.

 

Title: Please consider replacing it with "Physiological, cellular and transcriptomic analyses provide insights into the tolerance response of Arundo donax to waterlogging stress".

 

Materials and Methods

Lines 171-172: Was the experiment done under greenhouse conditions? Mercury lamps, daylight LED lamps or filtered sunlight provided the PPFD? Please provide complete information.

Lines 182-193: In section 2.3, all the mentioned parameters are biochemical. What about the physiological parameters?

 

Discussion and Conclusions

How the results are presented and discussed still does not align with the intended model.

The discussion should prompt an in-depth analysis of the model. To accomplish this, it is essential to thoroughly review and address previous and current results about each metabolic and signalling pathway included in the model.

Does starch or sucrose hydrolysis (produced by photosynthesis) provide glucose?

The waterlogging stress does not affect photosynthesis?

Have the authors explored other stress-induced pathways, such as lactate fermentation? Also, did the transcriptomic profile indicate differential expression of genes related to gluconeogenesis?

 

The authors have included tolerance responses to oxidative stress in the model but have not analyzed the effects on the electron transport chain (respiration). Are there alternative oxidases?

Comments on the Quality of English Language

It is necessary to revise the English grammar in the whole manuscript thoroughly.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The Authors reasonably responded to all the questions posed and made the corresponding changes which improved the quality of the Manuscript.

 

However, not all the amendments done are for good, so the Reviewer suggests to implement minor revisions.

 

1) Still the language is not ideal.

E.g. Figure S1 Morphological characteristics of roots in A. donax at different stages (0, 5, 15, 30, and 45 days) by the waterlogging stress

 

At different stages of waterlogging stress or a similar version, not by.

 

2)

 Abstract: Arundo donax is widely used as an ornamental plant in 17 landscape gardening because of preferable adaption to varying degrees 18 of waterlogged conditions.

 

“Preferable adaptation” doesn’t sound good, the previous version was better.

 

3)

 the 23 antioxidant enzymes (SOD, POD and CAT) in ARs also was greatly 24 enhanced after waterlogging.

 

The activity of antioxidant enzymes … or a similar version. The enzymes were not isolated and checked.

 

4)

 preferential and favorable 30 transcriptional response regulating

 

Favorable for which parameter? Doesn’t sound impressive.

 

5)

 indicated a preferential and favorable 30 transcriptional response regulating the AR development.

 

AR is better in full in abstract.

 

6)

 Taken 31 together, this study definitely advanced our knowledge about the 32 morphological, physiological and transcriptomic responses, and shed 33 new lights on the adaptive mechanism of A. donax under waterlogging 34 stress.

 

Definitely is not for abstract with the emotional form. Better: adaptive mechanisms, not mechanism.

 

7) In plants one ability to withstand waterlogging is the 59 formation of adventitious roots (ARs) and aerenchyma [10].

 

Language is not great.

 

8) Language problems still encountered. The fluorescence signal was observed on a laser scanning 215 confocal microscope (LSM880, ZEISS, Germany).

 

Using a laser scanning or a similar, not on.

 

9) 3.3 and figure 3, it’s better to provide the abbreviations in full.

 

10) =9 for all the remaining figures, better to provide more information for clarity in the figure legends to make the figures self-sufficient.

 

11) PYR/PYL is an acceptor of abscisic acid (ABA) signaling. 436 PP2C plays a negative regulatory role in the process of ABA 437 conduction.

 

Not an acceptor but receptor. Not conduction but signaling. Completely incorrect terms.

 

12) 3.6. Plant Hormone Signaling Pathways

 

Part of the story is for discussion section.

Comments on the Quality of English Language

Still needs to be improved.

Author Response

Please see the attachment

Author Response File: Author Response.pdf