Anatomical and Physiological Characteristics of Awn Development in Elymus nutans, an Important Forage Grass in Qinghai-Tibet Plateau

Round 1

Reviewer 1 Report

Review of Agronomy manuscript 2139500.

Relatively little research has been conducted on Elymus nutans, an important forage grass in the highlands of central Asia. The lemma awn of Triticeae species has played a critical role in the distributional and evolutionary success of this large group of plant species. A large number of genes are involved in the structure and size of awns.  For example, mutants at over 30 loci reduce awn length in barley. Hence, it is appropriate that a detail description of the awns of E. nutans be published.

The research characterizing the lemma awns or awns of E. nutans is well designed, carefully conducted and analyzed, and the manuscript is fairly well organized. However, the manuscript is poorly written and will require major changes before publication. The manuscript contains numerous grammatical errors and unusual uses of the English language. The introduction is poorly organized and not well focused on the central goal of the research. The discussion needs to consider primarily the significant differences between long and short awned genotypes. For example, does collection site have an influence on observed awn lengths?

Rejection of the manuscript is recommended primarily because the English used needs major changes. Below are some suggested improvements. Also, a number of details about experimental procedures need to clarified. The discussion does not consider large the difference presented in the results.

Only minor differences between long and short awned accessions; thus, the manuscript is largely a botanical description of the anatomical and biochemical traits of E. nutans awns.  This content may not be suitable for MDPI Agronomy.

 

Line 10: Suggested opening statements: “Awns are important structures of the inflorescence of many graminaceous crops. They have an active role in photosynthesis, transpiration, seed dispersal, and self-planting. Elymus nutans Griseb., which is an allohexaploid (2n = 6 = 42), self-pollinated, perennial, sparse clump grass of the tribe Triticeae, is an important native grass in Qinghai-Tibet Plateau. It exhibits considerable variation in awn length.”

Line 16: “awns” instead of “awn”

Line 18: Suggested statement changes: “The results showed that the cross sections of E. nutans awns had an acute triangle shape and structural similarities to wheat awns. In general, long awns grew faster than short awns at the heading stage, but no significant differences were found in awn length at heading, flowering, and maturity. There were no significant differences in the sizes of the barbs and stomata on the awn surfaces among the three accessions, however the long awned accession had more stomata than short awned accession at the four maturity stages.”

Line 33: Suggested statement: “Awns are needle like extensions at the top of glumes of Gramineae family members, which are involved in leaf metamorphosis and long-term adaptation and evolution.”

Line 37: This refers to lemma awns.  The most prominent awn in the spike is the lemma awn, which is developmental parallel to the leaf blade based on the phytomer concept generated for barley.

Line 40: All awns are slightly coiled, but they may appear straight.  Wetting and drying of the awn causes twisting and is part of the self-planting mechanism. Some species and mutants show much more twisting of awns. e.g Avena sp.

Line 85: “Based on previous awn length measurements, we choose the E. mutans accessions for further study:  …”

Line 88: “evenly on moist filter paper in 10-cm petri dishes.”

Line 90: “Seedling were grown under greenhouse conditions for eight weeks, and then vigorous seedlings were transplanted directly to the field.”  I assume seedlings were not preconditioned (exposed to outside conditions for a week to 10 days) before transplanting. At what time of the year were seedlings transplanted?

Table 1: The preferred designations for the PI accessions are PI 655186 and PI 619592 with a space between PI and the number.

Line 106: Were awns cut  or spikes harvested?

Line 109: “The inflorescence of E. nutans is a spike containing 20 to 30 spikelets. Most spikelets had 3 to 4 fertile florets with each having a long lemma awn.”

Line 112: “Awns were harvested from four spikelets near the center of each spike for further analyses.” Were awns cut only from primary florets?

Line 118: Awns have stomata and barbs as visual structures on the surface!

Line 125:  “The awn length of the primary floret of four central spikelets from each spike collected was measured.”

Line 305: Could reduced awn length and few barbs be an early step in the domestication of E. nutans?

Author Response

Dear colleague,

Thank you very much for your comments on our manuscript (agronomy-2139500). During this revision, we have carefully considered your suggestions, which are really useful for us to improve the manuscript. Our revisions as follows.

1. Line 10: Suggested opening statements: “Awns are important structures of the inflorescence of many graminaceous crops. They have an active role in photosynthesis, transpiration, seed dispersal, and self-planting. Elymus nutans, which is an allohexaploid (2n = 6 = 42), self-pollinated, perennial, sparse clump grass of the tribe Triticeae, is an important native grass in Qinghai-Tibet Plateau. It exhibits considerable variation in awn length.”

Answer: Thanks for your suggestion, we have replaced it, please see line 10-14.

2. Line 16: “awns” instead of “awn”

Answer: Thanks for your suggestion, we have corrected this mistake, please see line17.

3. Line 18: Suggested statement changes: “The results showed that the cross sections of  nutansawns had an acute triangle shape and structural similarities to wheat awns. In general, long awns grew faster than short awns at the heading stage, but no significant differences were found in awn length at heading, flowering, and maturity. There were no significant differences in the sizes of the barbs and stomata on the awn surfaces among the three accessions, however the long awned accession had more stomata than short awned accession at the four maturity stages.”

Answer: Thanks for your suggestion, we have taken your suggestion to replace it, please see line 18-23.

4. Line 33: Suggested statement: “Awns are needle like extensions at the top of glumes of Gramineae family members, which are involved in leaf metamorphosis and long-term adaptation and evolution.”

Answer: Thanks for your suggestion, we have taken your suggestion to replace it. We still add lemma, because awns are not only developed from glumes, please see line 32-34.

5. Line 37: This refers to lemma awns.  The most prominent awn in the spike is the lemma awn, which is developmental parallel to the leaf blade based on the phytomer concept generated for barley.

Answer: Thanks for your suggestion, we have changed “awns” to “lemma awns”, please see line 35-36.

6. Line 40: All awns are slightly coiled, but they may appear straight.  Wetting and drying of the awn causes twisting and is part of the self-planting mechanism. Some species and mutants show much more twisting of awns. e.g Avena

Answer: Thanks for your suggestion, we cited your words and revised the original text: All awns are slightly coiled, but they may appear straight. Awn’s twisting is caused by wetting and drying of the awn and plays an important role in self-planting mechanism. Generally, the long awns are more "curly" and the short are “straight”，please see line 38-42.

7. Line 85: “Based on previous awn length measurements, we choose the E. mutansaccessions for further study:  …”

Answer: Thanks for your suggestion, we have taken your suggestion to replace it, please see line 92-93.

8. Line 88: “evenly on moist filter paper in 10-cm petri dishes.”

Answer: We have taken your suggestion to replace it, please see line 95-96.

9. Line 90: “Seedling were grown under greenhouse conditions for eight weeks, and then vigorous seedlings were transplanted directly to the field.”  I assume seedlings were not preconditioned (exposed to outside conditions for a week to 10 days) before transplanting. At what time of the year were seedlings transplanted?

Answer: Yes. these seedlings were not preconditioned. We usually transplanted seedlings in Mid-April, the temparture is about 20 ℃, similar in Greenhouse. But we will take your suggestion before transplanting next time. Please see line 97-100.

10. Table 1: The preferred designations for the PI accessions are PI 655186 and PI 619592 with a space between PI and the number.

Answer: Thanks for your suggestion, we have checked our manuscript and we added spaces between PI and numbers.

11. Line 106: Were awns cut  or spikes harvested?

Answer: Sorry for this confusion. We cut spikes first, and then cut awns. According to your suggestion, we change "awns" to "spikes", please see line 113.

12. Line 109: “The inflorescence of E.nutans is a spike containing 20 to 30 spikelets. Most spikelets had 3 to 4 fertile florets with each having a long lemma awn.”

Answer: Thanks for your suggestion, we have taken your suggestion to replace it, please see line 117-118.

13. Line 112: “Awns were harvested from four spikelets near the center of each spike for further analyses.” Were awns cut only from primary florets?

Answer: Sorry for this confuion. We have revised: All awns of florets were harvested from four spikelets near the center of each spike for further analyses，please see line 121-122.

14. Line 118: Awns have stomata and barbs as visual structures on the surface!

Answer: Yes, we agree with you and already add this sentance in our text. Please see line 118-119.

15. Line 125:  “The awn length of the primary floret of four central spikelets from each spike collected was measured.”

Answer: Thanks for your suggestion, we have taken your suggestion to replace it, please see line 134-135.

16. Line 305: Could reduced awn length and few barbs be an early step in the domestication of  nutans?

Answer: This is good question, actually we agree with you, for example reducing awn length is also an early step of rice domestication. Next step we will sequence the whole genome of E. nutans and we further analysis the genetic variation among more different accessions with awn length variation.

17. Supplementary notes:

We have undergone extensive English revisions with the help of a native English speaker, then we have improved the introduction, research method and discussion section accoring your suggestion. The genes related to the awn development of Elymus nutans have not been reported, which is our further major reaserch objective. We will aim to reveal the mechanism of awn development from the aspects of phenotype, physiology and genetic regulation.

 

In general, we really appreciate your comments “Relatively little research has been conducted on Elymus nutans, an important forage grass in the highlands of central Asia. The lemma awn of Triticeae species has played a critical role in the distributional and evolutionary success of this large group of plant species....The research characterizing the lemma awns or awns of E. nutans is well designed, carefully conducted and analyzed, and the manuscript is fairly well organized”. We have tried our best to revise our manuscript carefully according to your and another reviewer’s valuable comments.  We wish this manuscript could be positively considered for publication in Agronomy.

 

Wish you all the best!

 

Yongsen Qiu and Wengang Xie

Lanzhou University

Reviewer 2 Report

General remarks to the authors:

I have read the manuscript with great interest as studies combining histology with biochemistry are rare and it would be a shame if it would not be published. I do find, however, that this work needs severe improvement before considering acceptance. A first concern is the language. Please have someone more familiar with the English language improve the text.

Introduction

The introduction should be more balanced. Whether awns may or may not present an advantage to cereal production is a matter of debate as highlighted in a recent review by Sanchez-Bragado et al. 2023, Trends in Plant Science. Information on possible correlations between awn length and ecological distribution in E. nutans would be helpful.

Results

Most descriptions would benefit if they would be more concise. Several Figures are redundant and should be removed. Regarding stomata number I wonder whether the higher values in the large awns are the consequence of the larger awns having a larger diameter (see remarks below).

Discussion

After gone through all the biochemical data, the only thing that sticks out is that a large awn has more content, needs more enzymes and more hormones than a small awn. All parameters are directly related to awn phenotype/growth which raises the question: where is the benefit for the seeds? Please elaborate on this.

 

Some specific remarks:

 

Line 13:  „greater“ please change into „large”

 

Line 26-27: delete “(CTK; SOD; POD)”

 

Line 27-29: “Therefore …. E. nutans” suggest to replace it with:

“The results obtained provide a sound basis for future research on the molecular mechanisms of awn development in E. nutans.”

 

Line 45-51:

The cells in the short awns are smaller, and most of them occupy in the middle.

I do not understand: are the awns smaller because the cells are smaller?

 

Rye (Secale cereale) and its close relatives lack sclerenchyma cells whereas; other species including 46 wheat and barley have parenchyma cells on both sides of awns that occupy a large part of the cross section of awns [4].

Rye has sclerenchymal cells but lacks parenchymal cells, change this into:

“The awns of rye (Secale cereale) and its close relatives lack parenchymal cells whereas in species like wheat and barley parenchymal cells make up a large part of awn tissue [4].”

 

 On the two sides of the distal end of awns, the stomata are distributed in bands. In addition, large numbers of stomata are arranged in the basal part 49 of awns as compared to middle and upper part [10]. The cross section of awns in barley, 50 wheat, and oat (Avena sativa) is triangular, with three vascular bundles and two thick walled tissues, which may contribute to photosynthesis [9].

Change into:

On the abaxial side of the awn two bands of stomata are present located on either side of the longitudinal axis. Stomata density is highest in the basal part of the awn. In barley, wheat and oat (Avena sativa) awns are triangular in diameter containing one median and two lateral veins separated by two bands of chlorenchymal tissue (9).

 

Line 64: Who is “Fabrice”? I guess it is an author but there is no information to be found.

 

Line 67: this means that de-awning reduces spikelet abortion?

 

Line 87-91: What happened between petri-dish incubation and transplantation into the field? Should it not be something like:

“To initiate germination seeds were laid out on moist filtering paper in 10 cm petri dishes. After …?... days young seedlings were transferred to …. ? ….and grown under greenhouse conditions for another 8 weeks after which they were transplanted into the field.”

 

Line 126-128: All awn tissues were collected from each inflorescence at four growth stages. The cut awns were stored in liquid  nitrogen, and then moved to the - 80 ℃ refrigerator for storage.

Please change into:

“After freezing in liquid nitrogen, isolated awns were stored in a -80°C refrigerator.”

 

Line 146-147: In the length of 2mm, the number of protruding thorns at four stages of 3 accessions was presented in Table 2.

Please specify from what area of the awn the samples were collected!

 

Line 156-157: Stomata of awns were less in booting stage, and the number of stomata in other stages was not different.

Change into: “In all accessions stomata number in awns did not increase after heading stage.”

 

Table 2:  Please delete as it is in essence a duplication of Fig. 4.

 

Table 3:  Please delete as it is in essence a duplication of Fig. 5. 

 

Fig. 6. The SEM image is far too unclear: please replace it with a light microscopy recording for which you can use hand-made transversal section through an awn. More important: given that you have three accessions with awns of different length, and given that these awns have different densities (?) of stomata, you should include awn transverse sections of all three accessions. Differences in diameter may explain the difference in stomata number.

 

Fig. 7. I would prefer to delete the figure altogether since the individual awns are too small for a good comparison and because the real data are given in Figure 8. If you insist on keeping Fig. 7, then have one awn per stage, per line as that makes it easier to see differences.

 

Line 195-200

This paragraph can be summarized as follows:

 “In all three accessions examined, awn length reaches it final expansion at heading stage (Fig. 8)”.

All other information in this paragraph is irrelevant.

 

Fig. 8. Please use the same style of bar diagrams as in Fig. 2 and Fig. 5. This makes it much easier to compare the data.

 

3.3. The difference of plant hormone activity in awns at four different stages

Line 208-248

This section should be shortened to let the information stand out better.

 

 

Line 298-320. General remarks to Discussion 4.1.

In its present form this paragraph can be deleted without affecting the story. It is general textbook information concerning the role of awns in seed dispersal and photosynthesis. If the authors want to keep it, I would suggest that they address following three remarks:

1)      In barley there is no relation between awn length and the presence/absence of thorns (smooth and rough awns). Working with three lines as the authors do, I find it premature to attach any significance to the observation long and thorny versus small and thornless.

2)      Awns have photosynthetic capacity but in barley this feature only plays a role in plants growing under harse conditions. In moderate climates awns have no clear advantage over awnless barley.

3)      I do not know of any breeding programms in cereals that use awns as a trait, please comment on this.

 

Line 322-324: Phytohormones are micro organic substances which can significantly regulate the growth and development of plants. They are endogenous, mobile and highly active substances with regulatory effects at low concentrations [25].

Please delete, this is basic knowledge all interested readers should be more than familiar with.

 

Line 340-342: Cao's research shows that the ABA content would increase only when the peach fruit entered the senescence period, and the ABA content was higher than that of ETH in the process of ripening and senescence.

Please change into: “Investigating peaches, Cao [30] observed that the ABA content of the fruit would rise during senescence and remain higher than that of EHT during both ripening and senescence.”

 

Line 372-373: CE and PG are two major degradation enzymes of plant cell wall. CE and PG mainly involved in the degradation of cellulose and pectin in cell wall respectively [36].

Please change into: “CE and PG are two major cell wall degrading enzymes, involved in the breakdown of cellulose and pectin, respectively [36].”

 

Line 387: and POD was more closely related to awns development.

It looks like a repeat of the line before so please delete this or rewrite.

Author Response

Dear colleague,

Thank you very much for your comments on our manuscript (agronomy-2139500). During this revision, we have carefully considered your suggestions, which are really useful for us to improve the manuscript. Our revisions as follows.

1. Introduction

The introduction should be more balanced. Whether awns may or may not present an advantage to cereal production is a matter of debate as highlighted in a recent review by Sanchez-Bragado et al. 2023, Trends in Plant Science. Information on possible correlations between awn length and ecological distribution in E. nutans would be helpful.

Results

Most descriptions would benefit if they would be more concise. Several Figures are redundant and should be removed. Regarding stomata number I wonder whether the higher values in the large awns are the consequence of the larger awns having a larger diameter (see remarks below).

Discussion

After gone through all the biochemical data, the only thing that sticks out is that a large awn has more content, needs more enzymes and more hormones than a small awn. All parameters are directly related to awn phenotype/growth which raises the question: where is the benefit for the seeds? Please elaborate on this.

Answer: The impact of awns on grain seed yield is controversial. According to your opinion, we have made a supplement to the introduction. This paper mainly focuses on the physiological basis of awn development. We have not measured the changes of hormones and enzymes in seeds during development, so we cannot highlight the impact of awns on seeds from a physiological perspective. We believe that even if we measure the changes of hormones and enzymes in seeds, we cannot explain the contribution of awns. We do not know which part or tissue these hormones and enzymes are produced, and whether they are transported from awns to seeds or from seeds to awn? This may complicate the problem. The specific effects of awn on seeds can be observed by other methods, such as the effects of awns removal on seeds and the application of isotopes. Please see line 69-75.

2. Line 13:  “greater” please change into “large”.

Answer: Thanks for your suggestion, we have revised the whole sentence, please see line 13-14.

3. Line 26-27: delete “(CTK; SOD; POD)”

Answer: Thanks for your suggestion, we have deleted “(CTK; SOD; POD)”, please see line 27.

4. Line 27-29: “Therefore …. E. nutans” suggest to replace it with: “The results obtained provide a sound basis for future research on the molecular mechanisms of awn development in E.nutans.”

Answer: We have taken your suggestion to replace it, please see line 27-28.

5. Line 45-51:The cells in the short awns are smaller, and most of them occupy in the middle. I do not understand: are the awns smaller because the cells are smaller?

Answer: There is no causal relationship between small cells and short awns. We just say that cells in short awns are usually small, please see line 46.

6. Line 45-51:Rye (Secale cereale) and its close relatives lack sclerenchyma cells whereas; other species including wheat and barley have parenchyma cells on both sides of awns that occupy a large part of the cross section of awns [4].

Rye has sclerenchymal cells but lacks parenchymal cells, change this into:

“The awns of rye (Secale cereale) and its close relatives lack parenchymal cells whereas in species like wheat and barley parenchymal cells make up a large part of awn tissue [4].”

Answer: We have taken your suggestion to replace it, please see line 46-48.

7. Line 45-51:On the two sides of the distal end of awns, the stomata are distributed in bands. In addition, large numbers of stomata are arranged in the basal part 49 of awns as compared to middle and upper part [10]. The cross section of awns in barley, wheat, and oat (Avena sativa) is triangular, with three vascular bundles and two thick walled tissues, which may contribute to photosynthesis [9].

Change into:

On the abaxial side of the awn two bands of stomata are present located on either side of the longitudinal axis. Stomata density is highest in the basal part of the awn. In barley, wheat and oat (Avena sativa) awns are triangular in diameter containing one median and two lateral veins separated by two bands of chlorenchymal tissue (9).

Answer: We have taken your suggestion to replace it, please see line 48-52.

8. Line 64:Who is “Fabrice”? I guess it is an author but there is no information to be found.

Answer: Sorry, we have made changes. The author name is Fabrice Ntakirutimana, please see line 64.

9. Line 67:this means that de-awning reduces spikelet abortion?

Answer: Removing the awn will reduce seed shattering degree, so the number of seeds on the spikelet will increase at the seed maturity stage, please see line 67.

10. Line 87-91:What happened between petri-dish incubation and transplantation into the field? Should it not be something like:

“To initiate germination seeds were laid out on moist filtering paper in 10 cm petri dishes. After …?... days young seedlings were transferred to …. ? ….and grown under greenhouse conditions for another 8 weeks after which they were transplanted into the field.”

Answer: Thanks for your suggestion. We have revised this part. To initiate germination, healthy, plump, and uniform size seeds were selected from each accession and placed evenly on moist filter paper in 10-cm petri dishes. They were incubated and germinated in an incubator at a constant temperature 25◦C with 12:12-h light/dark regime. After 10 days young seedlings were transferred to pots with 20 cm in diameter and grown under greenhouse conditions until they reached 8 weeks old, then healthy seedlings were selected and transplanted to the field in Mid-April.

11. Line 126-128: All awn tissues were collected from each inflorescence at four growth stages. The cut awns were stored in liquid  nitrogen, and then moved to the - 80 ℃ refrigerator for storage.

Please change into:

“After freezing in liquid nitrogen, isolated awns were stored in a -80°C refrigerator.”

Answer: We have taken your suggestion to replace it, please see line 135-136.

12. Line 146-147: In the length of 2mm, the number of protruding thorns at four stages of 3 accessions was presented in Table 2.

Please specify from what area of the awn the samples were collected!

Answer: We have added that“the samples were collected from the central area of awns”, please see line 155-156.

13. Line 156-157: Stomata of awns were less in booting stage, and the number of stomata in other stages was not different.

Change into: “In all accessions stomata number in awns did not increase after heading stage.”

Answer: We have taken your suggestion to replace it, please see line 163-164.

14. Table 2: Please delete as it is in essence a duplication of Fig. 4.

Answer: Table 2 presented the number of protruding thorns within the awn length of 2 mm at four stages of 3 accessions. But Fig. 4 showed the difference of stomal length and width in awns of three accessions at four developmental stages. Therefore table and figure can complement each other, we kept both in revised version.

15. Table 3: Please delete as it is in essence a duplication of Fig. 5. 

Answer: Thanks for your suggestion, we have deleted table 3 and kept Fig.5 in revised version.

16. 6.The SEM image is far too unclear: please replace it with a light microscopy recording for which you can use hand-made transversal section through an awn. More important: given that you have three accessions with awns of different length, and given that these awns have different densities (?) of stomata, you should include awn transverse sections of all three accessions. Differences in diameter may explain the difference in stomata number.

Answer: We tried to use the frozen section method and then put it under a light microscope for observation, and the result was very bad. This picture is the most "clear" one we have selected from more than 100 electron microscope scanning pictures. Because the awn itself is very small, and only about 1mm of the cross section is taken for observation, this will lead to two most critical problems: the scanning angle is biased and the structure of the awn's cross section is easy to "collapse". In revised version we put three more clear pictures to show the anatomical structure of long, medium and short awn accession. Please see line 196.

17. 7.I would prefer to delete the figure altogether since the individual awns are too small for a good comparison and because the real data are given in Figure 8. If you insist on keeping Fig. 7, then have one awn per stage, per line as that makes it easier to see differences.

Answer: Thanks for your suggestion. I agree with you. We have deleted Fig. 7 in revised version.

18. Line 195-200

This paragraph can be summarized as follows:

“In all three accessions examined, awn length reaches it final expansion at heading stage (Fig. 8)”.

All other information in this paragraph is irrelevant. 

Fig. 8. Please use the same style of bar diagrams as in Fig. 2 and Fig. 5. This makes it much easier to compare the data. 

Answer: We have taken your suggestion to replace it, but we still want to add the comparative data. Because Figure 7 also showed that the growth rate of long awn from booting stage to heading stage is faster than that of short awn. In Figure 7, we did not make the style of Figure 2, because we do not need to compare their awn length. The awn length of the long awn must be longer than the short awn. We just need to see at what stage each awn length reaches the longest. Please see line 200-201.

19. 3. The difference of plant hormone activity in awns at four different stages

Line 208-248: This section should be shortened to let the information stand out better.

Answer: We have shorten this section: Please see line 213-237.

20. Line 298-320.General remarks to Discussion 4.1.

In its present form this paragraph can be deleted without affecting the story. It is general textbook information concerning the role of awns in seed dispersal and photosynthesis. If the authors want to keep it, I would suggest that they address following three remarks:

1)      In barley there is no relation between awn length and the presence/absence of thorns (smooth and rough awns). Working with three lines as the authors do, I find it premature to attach any significance to the observation long and thorny versus small and thornless.

2)      Awns have photosynthetic capacity but in barley this feature only plays a role in plants growing under harse conditions. In moderate climates awns have no clear advantage over awnless barley.

3)      I do not know of any breeding programms in cereals that use awns as a trait, please comment on this.

Answer: According to your opinion, we have revised this paragraph, please see line 296-314.

21. Line 322-324:Phytohormones are micro organic substances which can significantly regulate the growth and development of plants. They are endogenous, mobile and highly active substances with regulatory effects at low concentrations [25].

Please delete, this is basic knowledge all interested readers should be more than familiar with.

Answer: We have deleted this part.

22. Line 340-342:Cao's research shows that the ABA content would increase only when the peach fruit entered the senescence period, and the ABA content was higher than that of ETH in the process of ripening and senescence.

Please change into: “Investigating peaches, Cao [30] observed that the ABA content of the fruit would rise during senescence and remain higher than that of EHT during both ripening and senescence.”

Answer: We have taken your suggestion to replace it, please see line 326-328.

23. Line 372-373:CE and PG are two major degradation enzymes of plant cell wall. CE and PG mainly involved in the degradation of cellulose and pectin in cell wall respectively [36].

Please change into: “CE and PG are two major cell wall degrading enzymes, involved in the breakdown of cellulose and pectin, respectively [36].”

Answer: We have taken your suggestion to replace it, please see line 357-358.

24. Line 387:and POD was more closely related to awns development.

It looks like a repeat of the line before so please delete this or rewrite.

Answer: We have deleted it, please see line 371.

In general, we really appreciate your comments and kind help with language editing. To meet the language requirement of Agronomy, we also have undergone extensive English revisions with the help of a native English speaker. We have tried our best to revise our manuscript carefully according to your and another reviewer’s valuable comments. We wish this manuscript could be positively considered for publication in Agronomy.

 

Wish you all the best!

 

Yongsen Qiu and Wengang Xie

Lanzhou University

Round 2

Reviewer 1 Report

The manuscript is now acceptable for publication.

Author Response

Dear colleague,

Thank you very much for your comments on our manuscript (agronomy-2139500). 

We have revised the language of our manuscript，including grammar and spell problem.

Thanks!

Wengang Xie

Reviewer 2 Report

Dear authors,

I appreciate your efforts to improve your manuscript. Text and figures are a lot better now and I understand why you did not include light microscope images of transverse awn sections.

I still have some issues with the language though, but if the editors have no problems with it, I suggest your work can be accepted for publication.

One final remark: In Fig. 7 you have forgotten to number the individal graphs (A, B, C).

 

Author Response

Dear colleague,

Thank you very much for your comments on our manuscript (agronomy-2139500). Our revisions as follows.

1. One final remark: In Fig. 7 you have forgotten to number the individal graphs (A, B, C).

Answer: Thanks for your suggestion, We have added A, B and C, please see line 198.

We also revised the language of our manuscript, including grammar and spell problem.

We do appreciate your comments and help during the revision.

Cheers

Yongsen Qiu and Wengang Xie

Lanzhou University