Effects of Exogenous Trans-Zeatin and Lovastatin on Abortion of Small Seeds in ‘Dawuxing’ Loquat (Eriobotrya Japonica Lindl.)

Round 1

Reviewer 1 Report

Manuscript Number: agriculture-1164532

This reviewer carefully read and revised the ms ID: agriculture-1164532 entitled “Effects of exogenous trans-Zeatin and Lovastatin on abortion of small seeds in ‘Dawuxing’ Loquat (Eriobotrya japonica Lindl.)”. This work is of interest in the field of rescuing new germplasm resources of loquat. Loquat small seeds are aborted during the seed development. In this study, the fruits of ‘Dawuxing’ loquat were treated by the cytokinin trans-zeatin and the cytokinin inhibitor lovastatin. Overall, the results showed that exogenous trans-zeatin significantly increased the metabolism of small seeds by monitoring morphological indexes and the levels of soluble sugars and starch, peroxidase and superoxide dismutase activities and gene expression. The same treatment increased endogenous trans-zeatin in the small seeds. Cytokinin dehydrogenase related genes were found to be down-regulated after trans-zeatin treatment. In conclusion, trans-zeatin treatment promoted the development of small loquat seeds during early stages of development, while cytokinin inhibitor (lovastatin) can significantly inhibit the development of small seeds.

I am very pleased to recommend the ms for publication in “Agriculture” after the suggested revisions. First, Authors have to highlight the scientific novelty and the importance of their discoveries. Then, in order to improve the ms, I recommend the authors to revise the text by removing some inaccuracies and checking for English language. The related suggestions are reported below:

Title page

L13: change “however” to “However”

L16: remove the plural in “inhibitors”

Intro

L45-46: check for English in “Cytokinins, as an important signaling mediator”, plural subject related to a singular term

L48: check for “effecetively”

L66: check for “significnatly”

L70: add reference/s and check for English in “However, the effect of exogenous cytokinin treatment on loquat seeds rarely been reported.”

L71: check for English in “trans-Zeatin (tZ)….. have been”

Methods

L84: remove comma

L101: change “ml” to “mL”

L103: use the corresponding RCF (g) instead of “rpm”

106-107: remove space in “1.  0 mL” and “0.  22”

L107. Remove “and”

L108: the analytes have to be detected, here use “subjected to analysis” instead of “detected”

L111: check for English verb in “The program of mobile phase gradient elution has been shown in table 1.”

L114: indicate the temperature used during the enzymes extraction

L115-116: clarify this sentence, in particular for “and the residue was added to 4 mL extract.”

L119: explain the acronym NBT = nitro blue tetrazolium

L123: remove the internal bracket

L129: check for “forever”, have to insert a specific time period

L132, 133: in Table 2-3, use the measure unit (µL) for the added volume

L137: insert company (and place) of Primer 5.0 software

L142, 143: check for English verb in “ have been shown”

L149-150: move these primers to Table 4

Results

L170: change “Table 6 to “Figure 1”, the same results are double repeated, please consider removing Table 6 (L180)

L172: remove “and”

L174: change “difference” to “different”, remove full comma before bracket

An error bar has to be insert for each data point of Figures 2, 3, 4, 5, 7, 9, and 11, as it is present for ckN in Fig. 11

L200: consider changing “ckA” to “ckN”

L206: insert Figure 3, remove “0-“ in “0-10 days”

L239. Insert the numeric information “5” and “7” for cytokinin oxidase/dehydrogenases in Table 4

L241. Insert “Figure 6” after “increased”

L248: change “ck” to “ckN”

L249: change “gene” to “genes”

Figures 6, 8, 10, 12 are not clearly legible; in addition, relative expression levels for each gene have to be reported as the fold increase relative to those of the untreated control, or please explain the alternative in this case with two controls (ckA and ckN)

L260: insert Figure 7

L261: insert “(ckA)” after “treatment”

L266: insert “(ckN)” after “treatment”

L278: in Figure 9, check for measure unit “POD activity (µ/(g)”

L283-284: remove “slowly” and change “10” to “15”

L299. Change “genes” to “gene”

L308: change “10” to “15”

L309: change “various” to “a”

Discussion

L319: consider moving “limited” before “availability”, and changing “nutrition” to “nutrients”

L346: check for English in “significantly increased fruit growth”, consider changing “increased” to “to increase”

L356-358: better explain the concept, is there consistency or not?

L364-367: describe the importance in this context by inserting references, or consider shorting this part

Discussion should be finalized to put together the results to support the initial idea, please consider this important aspect remodeling the final part of this section.

However, in this section, Authors have to highlight the scientific novelty and the importance of their discoveries for the rescue of new germplasm resources of loquat.

Author Response

Dear reviewer，

  Thank you vey much for your constructive comments! The point-by-point responses are listed in the attachment. Please see the attachment. Thank you very much!

Author Response File: Author Response.docx

Reviewer 2 Report

The paper “ Effects of exogenous trans-zeatin and lovastatin on abortion of small seeds in ‘Dawuxing’ loquat (Eriobotrya japonica Lindl.)” deals with an interesting topic. Unfortunately, I have many reservations about the written manuscript.

1) The paper was written very poorly. The text was composed of unrelated sentences, especially at the beginning of the manuscript, and requires linguistic correction as well. The authors of the article should pay attention to the instruction for authors prepared by the journal.

2) Please note the names of the reagents or proteins. Lovastatin and peroxidase should be lowercase.

3) The statement from the abstract: "...this study analyzed the effect of exogenous cytokinins on abortion of small loquat seeds, thereby laying a foundation for the research on embryo rescue, and thus providing an important idea and basis for the expansion and innovation of loquat germplasm resources using small seed breeding approaches.", is incomprehensible to me. What can the authors' research contribute to plant breeding? And what does the phrase mean "the expansion and innovation of loquat germplasm resources"?

4) Latin names of species should be italicized.

5) The article lacks information on how the fruit was sprayed and what was the weather at that time.

6) The spin speed should be reported as a g value.

7) I would suggest including the information from tables 1 - 5 in a supplement.

8) Sections 2.5; 2.6 and 2.7 in materials and methods can be combined into a single section.

9) Table 6 lacks the unit of measure and suggests writing in a smaller unit to avoid so many numbers.

10) The methods used by the authors to determine the content of sugar, protein, starch, and activity of antioxidant enzymes have been insufficiently described.

11) The discussion of the results obtained is superficial and should be completed.

Author Response

Dear reviewer，

  Thank you vey much for your constructive comments! The point-by-point responses are listed in the attachment. Please see the attachment. Thank you very much!

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I wanted to thank the authors of the manuscript for including my remarks. However, I have a few more comments:

1) The sentence "This study aimed to analyze the potential effects of exogenous cytokinin on abortion of small seeds of loquat, and cytokinin (20 mg/L trans-zeatin) as well as cytokinin inhibitor (19 mg/L lovastatin) were sprayed on the fruits of ‘Dawuxing’ loquat during an early stage of fruit expansion" (lines 16-19) is too long, not understandable and you have to divide it.

2) "...and the findings thus establish a foundation for the future research on embryo rescue in loquat" (lines 33-36; 439-440) In the summary, there is information about the embryo rescue technique, but it appears suddenly, it is difficult to link this technique with the results obtained by the authors.

3) "In March 2019, the fruit of ‘Dawuxing’ was sprayed with 20 mg/L trans88 zeatin(CAS: 1637-39-4), 60 mg/L lovastatin (CAS：75330-75-5) and clean water(CK) in clear days without windy conditions. After the whole surface of fruit was soaked for three times with hormones (once a day), the treated fruits were bagged in kraft paper bags. " (lines 88-90) So how was it with this fruit treating? Were they sprayed or soaked? If the plants are sprayed in the field, the following information is very important: temperature, cloudiness, rainfall, wind and time of day - this determines the drying or washing away of the working liquid and the possibility of its penetration into the plant.

4) In the description of the measurement of the concentration of sugars, proteins and starch, there is no information about the concentration of the solutions used: anthrone-ethyl acetate and Coomassie brilliant blue G 250 (this reagent is in powder form so the authors could not use pure 15ml reagent). What reagents were the standard for the determination of the calibration curves? Also, there is no information about the units given in tables 2, 3 and 4. The units given in the tables are completely incomprehensible. The same problem with units applies to measuring the concentration of zeatin.

5) All the analysis procedures to which the authors refer to publication no. 24 have been described much earlier by other authors and therefore the description of the methods in the manuscript should refer to the original publications.

6) In the descriptions of the methods of analyzing the activity of antioxidant enzymes, there is no information about the units in which the activity of enzymes was expressed. (lines 143-159).

7) In the Methods, the description of the analyzes carried out lacks information on the number of repetitions.

8) In Table 1, the value of SD has not been changed after converting the units from grams to milligrams.

9) In the description under Table 1, the sentence "The weight of per aborted small seeds is represented by g" is wrong and unnecessary.

10) The captions under the charts should contain information about the performed statistical analysis.

11) The Latin name of the species consists of two words, the first of which is written with a capital letter and the other with a lower case letter. (lines: 360; 361; 374; 380)

12) "Antioxidant enzymes such as SOD, POD, and CAT which are considered to be the main protective enzymes could remove the ROS and scavenge different free radicals." (lines 422-424) Please check the information that these enzymes also remove free radicals other than ROS. Only peroxidases can do this. SOD catalyzes the dismutation of the superoxide radical. CAT catalyzes the decomposition of hydrogen peroxide.

Author Response

Dear reviewer，

  Thank you vey much for your constructive comments! The point-by-point responses are listed in the attachment. Please see the attachment. Thank you very much!

Author Response File: Author Response.docx