Exogenous Phytohormones: Effects on Lettuce Photosynthesis, Antioxidant Response and Growth

Round 1

Reviewer 1 Report

 

 

Manuscript ID: Horticulture-2429846

Title: Exogenous phytohormones: effect on lettuce photosynthetic, antioxidant response and growth

In this paper, the authors explore the effects determined by the addition of phytohormone pairs when growing lettuce in pots.

The topic is well introduced as is the purpose and originality of the study. On the contrary, the description of the experimental plan has several gaps and numerous details need to be added for an accurate evaluation. The choice made about the statistical representation of the data also does not help to better understand the significant differences outlined within the Results section, where some statements are not in line with the reference graphs. This is naturally also reflected in the Discussion, which consequently needs to be revised.

Below are some points where action is needed:

 

Lines 31-33: the sentence is incomplete

Lines 84-85: "Chosen concentrations of phythormones" it is an important aspect
of this study and therefore it is necessary to spend a few more sentences on the reasons
for these choices.In Materials and Methods, the sections are numbered incorrectly, as the first number
is 4 instead of 2.

Lines 94-105: Did the pots contain different numbers of plants (5-7)?  The growing conditions used should be completed with a description of the characteristics of the peat used, and the cultivation practices of fertilisation, irrigation and pest defence. The experimental design adopted and the elementary plot are not reported. How were the pots arranged? It is unclear how the application of treatments was carried out; was the dose administered calculated as volume per plant or per surface area? what are the reasons for using an equal quantity ratio of 1 per phytohormone pair? what are sampling methods for lettuce biomass analyses? Finally, missing a description of the variety of lettuce used, which physiological stage does BBCH 12-13 refer to? what is the reason for this? bibliographical reference?

Line 148: the word "basil" must have been typed in by mistake

In Figure 3b, ABTS and DPPH are expressed, in a strange way, as millimolar concentration per gram "mM TE g^-1" (without any indication of whether fresh or dry weight), whereas in Materials and Methods section 4.5 (indeed 2.5) the authors report that ABTS and DPPH are expressed more correctly as micromol TE g^-1 F.W.

In Results, in different points there is no correspondence between the statements reported and the data represented in the figures and in particular the statistical significance of these data (see Line197-198,206-207,209-210, 216-217). The way of indicating significant differences between averages with an asterisk and not with letters does not clearly represent the comparison between them.

Line 190: "... and salicylic acids..." in combination with which other phytohormone? Line 191 " The same treatment..." Which ? Line 196: check the reference figure Line 197: add reference figure Line 218: delete GA (repeated twice) Line 220: " ....however no combinations showed significant effect on dry weight of the
plant ". Isn't that strange considering the previous statement? How does it fold?

Lines 232-239: In the results, the description of what emerged from the PCA analysis does not seem consistent with the PCA graph shown in Figure 4a.

The Results and Discussion sections should be reviewed on the basis of the previous observations.

Author Response

Dear Reviewer,

Thank you for all your comments and suggestions. Please, see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript reports the effect of exogenous phytohormones on lettuce photosynthesis,  antioxidant response, and growth.

Authors should moderately revise the text.

Main notes are due to a need for the moderate grammatical check of text (for the English language and style by professionals or editing companies).

It would be better to slightly change the title 'Exogenous phytohormones: effect on lettuce photosyntheticsis,  antioxidant response and growth'

The word 'biostimulant' should be written the same everywhere in the text, not separately 'bio stimulant'

It is worth adding the Latin name of the plant (Lactuca sativa instead of lettuce) in the list of keywords

It is also worth adding the Latin name of this plant (Lactuca sativa) after the words 'on lettuce antioxidant (….) – line 12.

line 15: Abbreviations 'BBCH' should be deciphered

line 15: 'was were applied'

line 17: 'two stress hormones lead led to'

line 24: 'factors often negatively influences'

 

line 335: 'to understand the positive and negative'

line 337: 'can give a positive outcoms'

line 340: 'and antioxidant responses, '

The purpose of the work is not clearly stated and should be corrected (lines 86-91).

The Introduction and Discussion sections could be slightly extended using the articles added to Bibliography published recently (2019-2023 years).

Main notes are due to a need for the moderate grammatical check of text (for the English language and style by professionals or editing companies).

Author Response

Dear Reviewer,

Thank you for all your comments and suggestions. Please, see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

This paper studied the effect of phytohormone-based bio stimulants on lettuce photosynthetic, antioxidant response and growth.

This research is significant. Yet, the current quality of manuscripts does not match demand and must be enhanced.

Abstract:

1. in the end, please add the implication of this research briefly.

Introduction：

2. The regulatory role of different phytohormones is described here, however, this is not enough to reveal research gaps. Is there any existing research on the interaction between two or more?

Materials and methods:

3. Where is the location of the test site? Or the location of the greenhouse.

4. What about the experiment time?

5. In order to improve the reproducibility of the experiment, more information is needed. Such as the material, and height of the vessel. Is there no growing medium or soil in the vessel?

6. For PCA and Clustering, what software is used for plotting?

Results:

7. For Fig. 4b, all treatments came to two main clusters. What is the meaning of this finding? It seems that there is no description on this in "Discussion".

Please check for some typos and grammar. For example,

5 days after application (line 104): "5" should be revised to "five"; a space needs to be added in front of this sentence.

The height of the plant is measured with...(line 108): "is" should be revised to "was".

 

Author Response

Dear Reviewer,

Thank you for all your comments and suggestions. Please, see the attachment.

Author Response File: Author Response.docx

Reviewer 4 Report

Dear authors

After reading and analyzing your manuscript, I give you the following remarks:

 

1)      The title is correct.

2)      The introduction part is very long in addition the authors have developed the results a lot, normally the introduction is generally having the main functions of presenting the subject and announcing the plan of a written document.

3)      In the material and methods part, I prefer to add images of the palm and the greenhouse.

4)      Also, it is possible to add physico-chemical analyzes to characterize the plant, pH, Ash, heavy metals, oraganic matter, humidity, .......

5)      justify your discussion; Our findings show that combinations of GA with other hormones haven't a meaningful impact on photosynthetic rate (Fig 1. A).

6)      the result and discussion part are perfect but add images of your sample work.

7)      for the conclusion it is preferable to add a perspective of this work.

Best Regards

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you for all your comments and suggestions. Please, see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

 

Manuscript ID: Horticulture-2429846

 

Title: Exogenous phytohormones: effect on lettuce photosynthetic,

antioxidant response and growth

The second version of the manuscript is only slightly improved on the first. With regret
I have noticed that many of my observations have not been taken into consideration.
The modifications or the answers given to some of them have not proved satisfactory
in clarifying the questions posed. I report below the points that need definitive
clarification, essential to be able to recommend the publication of the article in this
journal. For clarity, I add my comments point by point directly to the cover letter sent by the
authors: in green the answers of the authors, in black my comments on the first version,
in red my comments on the second version.   Dear Reviewer,

We would like to thank for taking the time and effort necessary to review the manuscript. We sincerely appreciate all valuable comments and suggestions, which helped us to improve the quality of the paper. Changes were applied in the text according to Your comments.

Lines 31-33: the sentence is incomplete

Statement is corrected  

Lines 35:  " resultsing" ????

Lines 84-85: "Chosen concentrations of phythormones" it is an important aspect
of this study and therefore it is necessary to spend a few more sentences on the reasons for these choices

 

Rates of 30 mg l-¹ was chosen from previous experiments and results were the basis for this research. These results aren’t published yet but were presented at IHC 2022 and SAIH 2022 conferences. In our experiment we decided to use on-time foliar exogenous phytohormone application, because of rapid development of lettuce and the highest effectiveness can be reached at early stages of plant growth.

 

The results on the basis of which the choice of experimental procedure was made should be briefly reported. This information should be rewritten and included in the text. (Lines 91-92)

 

In Materials and Methods, the sections are numbered incorrectly, as the first number
is 4 instead of 2.

Numbers are corrected.

 

OK

Lines 94-105: Did the pots contain different numbers of plants (5-7)? 

Seed number was specified in methodology. After germination one pot contained 5 plants.

OK

The growing conditions used should be completed with a description of the characteristics of the peat used, and the cultivation practices of fertilisation, irrigation and pest defence.

According to good experiment practice of vegetative experience all management control was assured. Because plants were grown in a greenhouse under controlled environment, additional tools such as fertilization or pest defence was not needed. We added soil parameters in methodology section.

Growth in a greenhouse under controlled environment does not exclude the need for fertiliser inputs and plant protection measures. It is a different matter if the nutrients in the substrate are sufficient for the short development period of the experimental trial

Line 103: "sudétis" ??

The experimental design adopted and the elementary plot are not reported. How were the pots arranged?

The lettuce were seeded into 0,5l vessels, five plants in each and 18 vessels represented one treatment replication.

The answer is not clear.

“ 0,5l vessels, five plants in each”: Since it is stated in materials and methods that 5 plants are sampled and in the statistics section that the number of replications is 5, it can be deduced that each treatment is applied to a total number of 5 plants. It is also deduced that these 5 plants, representing the 5 replicates are in one pot.

These do not appear to be appropriate experimental conditions.

Otherwise describe more clearly

“18 vessels represented one treatment replication”.: It is not clear what it means.

It is unclear how the application of treatments was carried out; was the dose administered calculated as volume per plant or per surface area? what are the reasons for using an equal quantity ratio of 1 per phytohormone pair?

Based on previous results of separate hormone treatments we decided to use an equal quantity ration per phytohormone pair. As we mentioned earlier, this data is not published yet, but were presented in IHC 2022 and SAIH 2022 conferences.

As already mentioned, the results on the basis of which the choice of experimental procedure was made should be briefly reported.

This information should be rewritten and included in the text.

Line 112:113.  The authors report that the phytohormone solution was applied as volume per surface area (ml/m2). Therefore the number of plants per square metre should be indicated. Also, the number (2001 ml to 1 m 2) should be corrected, I think.

what are sampling methods for lettuce biomass analyses? Finally, missing a description of the variety of lettuce used, which physiological stage does BBCH 12-13 refer to? what is the reason for this? bibliographical reference?

For biochemical analysis material was freeze-dried and for dry biomass plants was determined by drying them in +70ºC for 48 h. This was corrected in methodology section.

BBCH 12-13 refer to seedling stage. In other terms when second leaf was unfolded. Because of rapid development it is a critical organogenesis stage for lettuce. Because of this, highest effectiveness can be reached when phytohormone-based biostimulants are applied at early stages of plant growth.

“BBCH 12-13 refer to seedling stage. In other terms when second leaf was unfolded”

A description of the developmental stage of the lettuce corresponding to BBCH 12-13, to which the treatment is applied, should be added in the text. So, according to Table 1, after 5 days the third and fourth leaves develop?

Line 125: "Papildyti žm sm " ????

Line 148: the word "basil" must have been typed in by mistake

Sentence was corrected.

OK

In Figure 3b, ABTS and DPPH are expressed, in a strange way, as millimolar concentration per gram "mM TE g^-1" (without any indication of whether fresh or dry weight), whereas in Materials and Methods section 4.5 (indeed 2.5) the authors report that ABTS and DPPH are expressed more correctly as micromol TE g^-1 F.W.

Figures and methodology was corrected.

In the modified text (lines 161 and line 167) and in fig 3b the units of measurement
with which the results of ABTS test and DPPH assay are indicated are still unclear and
different. We need to unify and clarify. What is meant by D in "micromol g^-1 DFW " ?
Why in the text is indicated as a unit of measurement for both tests? Typically, DPPH
test results are indicated as "micromol g-1 TE FW" where TE stands for Trolox
Equivalent.   In Results, in different points there is no correspondence between the statements
reported and the data represented in the figures and in particular the statistical
significance of these data (see Line197-198,206-207,209-210, 216-217). 
The way of indicating significant differences between averages with an asterisk and not
with letters does not clearly represent the comparison between them.  

Text was corrected according to suggestions.

  No changes have been made by the authors regarding my observation. Therefore
I repeat it in more detail: Line 214-215 (ex 197-198): the sentence is incorrect; even treatments with SA cause
a statistically significant decrease in the NPQ parameter (KIN+SA, IAA+SA).
GA+SA also causes the same effect even if the decrease is not statistically significant.
Only ABA+SA has the opposite effect. Line 223-224 (ex 206-207): the sentence is imprecise; the only value of the TPC
parameter statistically different from the control is, in fig 3a, GA+SA and therefore
it is the only one that can be commented Line 226-227 (ex 209-210): the sentence is imprecise; from fig 3c it can be seen that
the differences between the FRAP values ​​found for KIN+ABA, and GA+ABA and
the control are not statistically significant; on the other hand the treatment with
GA+SA decreases significantly from a statistical point of view from the control but
no comment is included in the text. Line 234-235 (ex 216-217): from table 1 the difference of leaf area between
GA + ABA and the control, although present, is not statistically significant. The same
applies to IAA+SA and ABA+SA, which are not highlighted in the text. On the other
hand, the statistically significant increase for GA+SA is evident, strangely not mentioned
in the text.

Line 190: "... and salicylic acids..." in combination with which other phytohormone? 

Again the authors did not clarify. In Line 207 (ex 190) there were no changes   Line 191 " The same treatment..." Which ? 

OK

Line 196: check the reference figure 

??

Line 197: add reference figure

??

Line 218: delete GA (repeated twice)

OK

Line 220: " ....however no combinations showed significant effect on dry weight of the plant ". Isn't that strange considering the previous statement? How does it fold

Uncommented observation

Lines 232-239: In the results, the description of what emerged from the PCA analysis does not seem consistent with the PCA graph shown in Figure 4a.

It is incorrect to add a table 4c inside the figure 4 like this; the caption of figure 4
needs to be corrected; delete or translate the sentence written in Lithuanian.

The comment to fig 4a in the text has not been corrected.

Figures 4a and 4c show that F1 and not F2, as stated by the authors, influenced the response of the antioxidant system.

  Line 368-371: the added sentence is unclear   Results and Discussion sections should be reviewed on the basis of the previous
observations.   I reiterate the need to review Results and Discussions on the basis of the above

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

We would like to thank You one more time for Your valuable insights reviewing the manuscript.  Changes were applied in the text according to Your comments.

Author Response File: Author Response.docx

Reviewer 4 Report

Dear authors 

nothing to declare

Best regards

Author Response

Dear Reviewer,

We would like to thank You one more time for Your valuable insights reviewing the manuscript.

Round 3

Reviewer 1 Report

The submitted article is partially improved from the second version. Many of my comments have been taken into account and the right changes have been made. Therefore, the experimental design is now clearer and appropriate for the purpose of the work. The changes reported in Results are consistent with my suggestions. However, a number of points remain unclear, which the authors do not answered.

 

Here are the points in need of change:

1. In "2.5. Antioxidant activity and total phenolic content".  The units of the DPPH and ABTS assays described in the article (lines 159-160 and 165-167) are still different from those in Fig. 3b. It is necessary to merge the test and the figure and, if necessary, to change the subsequent numbers and comments. In addition, the expression mM TE g^-1 is incorrect (it indicates the trolox equivalent concentration x gram of sample); the correct unit is micromol TE g^-1.

2. In the authors' response letter to report 2, there is no comment on my observation 'Figures 4a and 4c show that F1 and not F2, as stated by the authors, influenced the antioxidant system response'.     It is clear that all parameters linked to the antioxidant system (DPPH, ABTS, FRAP) as well as the Y(II) and ETR parameters are linked to the F1 factor and not F2. The comments linked to the explanation of the data presented in fig 4a,c  have to be revised (Line 255-258) and readjusted. Figure 4c appears in duplicate in the text.                                                                                                 

This also involves a change in what is written in Results (lines 345-347) which repeats the same statment.

3. In Results the authors made the appropriate corrections according to my suggestions (Lines 214-215, 223-224, 226-227,234-235). On the other hand, contrary to what was stated in the reply letter, the text of the Discussions remained substantially unchanged from the previous version. Therefore, some of the present statments are inaccurate:

Lines 272-279: the sentence " Results of the experiment indicate that treatment with GA + ABA signifiicantly increased average leaf area of a plant, which is an important productivity factor for a leafy vegetable."  is not supported by the results: treatment with GA+ABA does not produce statistically significant differences in this parameter compared to the control. Consequently, the next sentence also "Although there is information that ABA and GA interaction is antagonistic in germination processes [286], but significant increase in leaf area shows synergistic crosstalk between these two phytohormones occurs after germination processes passed."  needs to be modified

Lines 318-321 : is not true that "The combination of KIN and GA significantly increased the .......non-photochemical quenching."

Lines 339-341: "Our findings show that photochemistry results (Fig 2) of such treatment were significantly reduced and NPQ levels significantly increased, compared to control, which means that light is not used in an efficient way." It is not true that NPQ levels generally increase (this is only true for ABA+SA). Therefore, the sentence and its conclusion must be adjusted.

Author Response

Dear Reviewer,

Once again, thank You for Your valuable insights to our article. We corrected text according to Your suggestions. You will find changes in the attachement.

Author Response File: Author Response.docx