The Structure, Function, and Regulation of Starch Synthesis Enzymes SSIII with Emphasis on Maize

Round 1

Reviewer 1 Report

The English level presented in this manuscript is not completely up to the publication level yet. I suggest that the manuscript should have careful proofreading and language editing and can be re-submitted once the English level is improved to the publication level. Please read the Agronomy papers and follow the formatting, for example, the References.

Items to work on to improve, for example,

1. Fix grammar errors.
2. Figure resolution and font in the figures. Specially, Figure 3 text not readable.
3. Add figure title and legend to explain the figures.
4. There are two Figure 4! One is after Figure 5.
5. Conclusion should be brief and precise.

Author Response

Dear reviewer,

Thank you for your careful reading and positive comments and suggestions upon our manuscript. We have added a point-to-point response to your comments in the revised version of manuscript. We felt that after the incorporation of changes you suggested, manuscript quality has been greatly improved. We have highlighted all the changes we made in red color.

 

1. Fix grammar errors.

Thanks for your carefully checking! Our manuscript has been checked by a native English-speaking colleague and the manuscript has been carefully proofread for grammar, punctuation, and English language mistakes.

 

2. Figure resolution and font in the figures. Specially, Figure 3 text not readable.

Thanks for your suggestion! We have split and changed the layout of the previous Figure 3, and the Figure 3 and Figure 4 now have a better readability.

(P5, line 156 and P6, line162)

Figure 4. Tertiary structure prediction of SSIII genes, CBM25, GT5, and GT1 structural domains are highlighted in orange, red, and yellow colors separately. It can be seen that the spatial distribution of structural domains showed consistency. Consistent spatial distribution laterally reflects that the structural domains could play a central role in SSs

 

3. Add figure title and legend to explain the figures.

Thanks for your suggestion! We have made additional changes to the legend of each of the previous Figures, except for Figure 4. Transcriptional level regulation of SSIII (Line no. 340), which has been removed.

(P3, line 99; P5, line 157; P6, line 163; P7, line 195 and P8, line 239)

Figure 2. Structural features of maize amylopectin. (a) Pattern diagram of maize amylopectin in the wild type. The hexagons are indicating glucose molecule and the short horizontal lines indicating glycosidic bonds. (b) Pattern diagram of maize amylopectin in the Dull1 mutants. The hexagons are indicating glucose molecule and the short horizontal lines indicating glycosidic bonds. The Dull1 mutations lead to fewer long cluster on B chains, increasing amount of short chains and larger cluster in branches in amylopectin with more singly branched building blocks.

 

Figure 3.  Composition and distribution of domain structures and conserved motifs of SS proteins. Zea Mays (Zm), Oryza sativa (Os), and Arabidopsis thaliana (At) are shown as an example. The N-terminus is on the left side of the figure and C terminus is on the right side. The three functional structure domains GT, CBM, and CC are marked in black font, and the predicted conserved motifs are marked in white font. Detail gene list is in Supplemental table S1.

 

Figure 4. Tertiary structure prediction of SSIII genes, CBM25, GT5, and GT1 structural domains are highlighted in orange, red, and yellow colors separately. It can be seen that the spatial distribution of structural domains showed consistency. Consistent spatial distribution laterally reflects that the structural domains could play a central role in SSs function.

 

Figure 5. Expression level of SSIII genes in different tissues. In maize seed, the expression levels of SSIIIb-a and SSIIIb-b maintain a low level while there was a high level expression phase of SSIIIa from day 8 to 28, showing an overall trend of first increasing and then decreasing. In the embryo, the expression levels of SSIIIa, SSIIIb-a, and SSIIIb-b are found higher than those in the seeds. In the endosperm, the expression of SSIIIa is found to be dominant with the highest expression occurring from 12 to 26 DAP, and the expression showed an overall trend of increasing and then decreasing from 6 to 36 DAP. In addition, by analyzing the expression pattern in other different tissues of maize, it is found that the expression of SSIIIa is low in all other samples except in cob; SSIIIb-a expression is high in shoots and leaves; SSIIIb-b expression is high in cob and silk [44]. The data used for graphing are listed in the Supplemental table S2.

 

Figure 6. The network of interactions proven by curated databases or experiments between several key enzymes and SSIIIa in starch synthesis. Maize is shown as an example. Interaction network suggesting that SSIIIa can be involved in the process of starch biosynthesis and cellular glucan metabolism. The abbreviated gene name corresponds to its full length. SSIIIa: starch synthase IIIa; DPE2: 4-alpha-glucanotransferase DPE2; Agp2: ADP-glucose pyrophosphorylase2; SBE1: amylose extender 1; sh2: shrunken 2; GLG1: ADP glucose pyrophosphorylase small subunit leaf 1; waxy: Granule-bound starch synthase 1; bt2: Glucose-1-phosphate adenylyl transferase; AGP2: ADP-glucose pyrophosphorylase; sbe3: starch branching enzyme 3; sbe1: starch branching enzyme 1. Empty nodes: proteins of unknown 3D structure, filled nodes: known or predicted 3D structures. Genes and protein sequences list is in Supplemental table S3.

 

 

4. ‘There are two Figure 4! One is after Figure 5.’

Thanks for your carefully checking! We are very sorry and have verified the numbering of each Figures in the current manuscript.

 

5. ‘Conclusion should be brief and precise.’

Thanks for your suggestion! We have carefully revised the Concluding Remarks and Future Perspectives section to remove unnecessary parts, more accurately summarize the current research progress, and state the authors' outlook on the future direction of SSIII research.

(P12, line 383-434)

The biosynthesis of starch is a highly complex metabolic process that requires the synergistic action of multiple enzymes. Studies targeting its synthetic pathways have been a long-standing research hot spot, while there are still ongoing controversies and corrections [87]. Existing studies have shown that SSIII acts as the core and regulatory enzyme of maize starch synthase with several important functions including synthesizing highly ordered branched starch, influencing the structure of branched starch and catalyzing the formation of longer sugar chains.

Regarding its physicochemical properties SSIII found to be consisted of a specific transport peptide region. The structure is characterized with a Dull1-specific N-terminal region, a central region (homologous to other class III starch synthesis), and a C-terminal region containing a catalytic structural domain for a total of four functional regions. Several other structural domains including CC, GT5, GT1 and CBM25 have been identified in SSIII, however no direct studies have been conducted to the date to demostrate the functional link of the structural domains with the function of SSIII in starch synthesis. Despite the variations in the sequences the predicted tirtiary structures showed the consistency in the spatial distribution of the structural domains in each of the mentioned isoforms which have not be characterized yet. In terms of its optimal working conditions, only a preliminary study described the effect of temperature on SSIII is available. Considering the central position of SSIII in starch synthesis, further investigation of the optimal physiological conditions of SSIII could be significant for adjusting cultivation practices in maize.

The expression of SSIII found maximum during 10-30 DAP stages and expression of SSIIIa is found dominant in the endosperm. The expression of the starch biosynthetic enzyme significantly affects the starch content of the seeds in these stages suggesting SSIII could also effect on starch yield. The expression results can be utilized to the breeding work of transgenic maize varieties.

In relation to regulation of metabolic pathways several studies have shown that SSIII can be a "regulator" of other key enzymes for starch synthesis including SBE, other SS, and GBSS which act directly or indirectly to participating enzyme. Several reports suggested that SSIII is involved in both starch biosynthesis and cellular glucan metabolism. It is reported to perform several biological activities including glycogen (starch) synthesis activity, starch-binding activity and transferase activity for transferring hexose molecules. SSIII in multiple enzyme complexes with SBEs, SSs, ISA, PUL, PHO and other key enzymes for starch synthesis have been reported in maize and rice crops. However, the pathway of enzyme complex formation and the specific catalytic function of each part of the enzyme complex have not been characterized yet. Furthermore, several enzymes in a complex reported to be modified via phosphorylation and the exact fate of phosphorylation needed further clarifications. phosphorylation starch synthases are thought to be a common phenomenon [53] it can be speculated that SSIII can also modify via phosphorylation, since there is no direct evidence for the phosphorylation have been reported so far, further studies are needed.

Several key elements regulating starch synthesis have been identified in the promoter region of SSIII, however experimental verifications are missing. Further studies are needed to demonstrate the functional importance of the identified elements. Transgenic experiments on PDull1 have shown that PDull1 act as potential tool to improve crop yield. Transcription factors have been identified to regulate the starch synthesis process in plants It can be speculated transcription factors regulating SSII could be influenced by several signaling molecules including sucrose, ABA, miRNAs and set of several other interacting molecules. Considering the important regulatory role of transcription factors we need detection and identification of transcription factors, signaling molecules and miRNAs in future studies which could help to determine the underlying pathways of signaling molecules and miRNAs for the regulation of SSIII function by transcription factors.

 

6. Please read the Agronomy papers and follow the formatting, for example, the References.

Thanks for your carefully checking! We apologize for this and have made changes.

(P14, line 474)

 

Author Response File: Author Response.pdf

Reviewer 2 Report

I have carefully read the review entitled “The Structure, Function, and Regulation of Starch Synthesis 2 Enzymes SSIII with Emphasis on Maize” by Yu et al. I have seen that the such review is not available on web considering SSIII and maize relationship but this article is not at all presented in an understandable way. It is hard to establish the link between the various sections. Article is having low readability both text and figures. Even tabular content is not supplemented in any of the section. Table is important ingredient to make the review article impactful.  Therefore, I am not recommending the review article for publication in “Agronomy”. I am suggesting few points for allowing authors to rethink and improve the quality of the manuscript:

In introductory section authors must say the importance of maize with respect to overall starch production and comparing it with other crops such as rice, potato etc. Authors must also clearly highlight the novelty of the review in the last para of the introductory section and report how the current article is different from the previous article published recently. Discussion part need further improvement. Moreover, cited literature contains many old references which need to be updated in order to improve the quality of the manuscript. I have observed that manuscript is rarely cited with recent references (2019-2022).

Further, authors view points are missing from the manuscript. Author must add their view point at end of each section/subsection.

Figure 3 is not at all clear. Authors must improve it to make it more readable.

Figure 4 must be explained with detailed legend (Expression level of SSIII genes in different samples).

Figure 4. Transcriptional level regulation of SSIII (Line no. 340): this figure need to be revised with details; how various transcription factors can regulate SSIII. Accordingly detailed legends must be given.

Numbering is wrongly mentioned in the figures. Authors needs to properly check and accordingly make changes in the text.

Numbering of section is also need to be checked. I dont know what is the significance of section 8.

Please improve the conclusion as per the exact findings of the review article. Current form of conclusion is very crude and need improvement.

English needs careful check. I have seen various mistakes. Even punctuations are not used in proper way.

In the current format review article will not add any kind of new information to the readers of the “foods” and also it will not able to create an impact to improve the standards of the journal. If authors can properly justify all the above-mentioned concerns, I am happy to re-review the manuscript.

Author Response

Response Letter to Reviewer

Dear reviewer,

Thank you for your careful reading and positive criticism and suggestions upon our manuscript. We have added a point-to-point response to your comments in the revised version of manuscript. We felt that after the incorporation of changes you suggested, manuscript quality has been greatly improved. We have highlighted all the changes we made in red color.

 

1. English needs careful check. I have seen various mistakes. Even punctuations are not used in proper way.

Thanks for your carefully checking! Our manuscript has been checked by a native English-speaking colleague and the manuscript has been carefully proofread for grammar, punctuation, and English language mistakes.

 

2. Figure 3 is not at all clear. Authors must improve it to make it more readable.

Thanks for your suggestion! We have split and changed the layout of the previous Figure 3, and the Figure 3 and Figure 4 now have a better readability.

(P5, line 156 and P6, line162)

line162)

Figure 4. Tertiary structure prediction of SSIII genes, CBM25, GT5, and GT1 structural domains are highlighted in orange, red, and yellow colors separately. It can be seen that the spatial distribution of structural domains showed consistency. Consistent spatial distribution laterally reflects that the structural domains could play a central role in SSs

 

3. Figure 4 must be explained with detailed legend (Expression level of SSIII genes in different samples).

Thanks for your suggestion! We have made additional changes to the legend of each of the previous figures. The previous “Figure 4. Expression level of SSIII genes in different samples” has been replaced with “Figure 5. Expression level of SSIII genes in different tissues” which is equipped with detailed figure legend.

(P7, line 195)

Figure 4. Tertiary structure prediction of SSIII genes, CBM25, GT5, and GT1 structural domains are highlighted in orange, red, and yellow colors separately. It can be seen that the spatial distribution of structural domains showed consistency. Consistent spatial distribution laterally reflects that the structural domains could play a central role in SSs function.

 

4. Numbering is wrongly mentioned in the figures. Authors needs to properly check and accordingly make changes in the text.

Thanks for your carefully checking! We are very sorry and have verified the numbering of each figure in the current manuscript.

(P2, line 61; P3, line 99; P5, line 157; P6, line 163; P7, line 195 and P8, line 239)

 

5. In introductory section authors must say the importance of maize with respect to overall starch production and comparing it with other crops such as rice, potato etc.

Thanks for your suggestion! We state the importance of maize in food crops and describe the importance of maize in the overall starch production by illustrating the extremely high content of starch in maize and the indispensable position of maize starch in the starch industry in the United States and Europe.

(P1, line 29, 30, 32-35)

Maize is one of the major food crop in the world and can be utilized as the resources for food, animal feed and biofuel [1, 2]. Starch is the primary storage form of carbohydrates and the second most abundant biopolymer next to cellulose [3]. It is one of the final product of photosynthesis and utilizes as an energy source in plants [4, 5]. In maize, starch is the main component accounting for about 75% of its total dry matter content [1]. The production of maize starch dominating the starch industry in the United States and European regions [6].

Starch can be divided into two types; transient starch and storage starch [7]. In photosynthetic tissues, transient starch accumulates in chloroplasts during the daytime. At night, it’s hydrolyzed to maltose and glucose (Glc) to support respiration and metabolism [8]. Storage starch is deposited in the specialized compartments (amyloplast) of the non-photosynthetic tissues (endosperm, tuber, and roots) [9]. Furthermore, it can be used not only as a key factor in determining the quality of food or feed products but also as a cost-effective biodegradable and renewable industrial raw material. It is widely used in the industries related to energy, chemicals, medicines, construction, and plastic production [10-12].

In maize endosperm, sucrose is the carbon source for starch synthesis which is transported to the cytoplasm and converted into ADP-glucose (ADPG) by cytosolic ADP-glucose pyrophosphorylase (AGPase). The ADPG is transported into amyloplast to form α-glucose-1-phosphate (G-1-P) which is converted into starch through a series of reactions [13]. Starch synthase III (SSIII) forms a complex with AGPase, pyruvate phosphate dikinase (PPDK), starch synthase IIa (SSIIa), starch branching enzyme IIa (SBEIIa), and starch branching enzyme IIb (SBEIIb) [13]. In the complex, SSIII is found as the largest molecular weight protein resides in a central position and links the phosphate transfer and starch synthesis [13] (Figure 1).

In summary, maize starch is dominating in the overall starch production and SSIII could significantly affect the starch production because of its central position in the enzymes complex system. Relationship between SSIII and maize starch biosynthesis has not yet been reported clearly. In this paper, we have studied the structure, function and regulation of SSIII. Moreover, physicochemical properties, expression, protein-level interactions and regulation at the transcriptional level have also been concluded.

 

6. Authors must also clearly highlight the novelty of the review in the last para of the introductory section and report how the current article is different from the previous article published recently.

Thanks for your suggestion! As the reviewer said “Such review is not available on web considering the relationship between SSIII and maize.”, so we cannot report how this article differs from the recently published previous article. However, in the last paragraph of the introduction section we describe the purpose of writing this paper, the current status of the research area reviewed in this paper and the main points reviewed in this paper.

(P2, line 54-59)

In summary, maize starch is dominating in the overall starch production and SSIII could significantly affect the starch production because of its central position in the enzymes complex system. Relationship between SSIII and maize starch biosynthesis has not yet been reported clearly. In this paper, we have studied the structure, function and regulation of SSIII. Moreover, physicochemical properties, expression, protein-level interactions and regulation at the transcriptional level have also been concluded.

 

7. I have observed that manuscript is rarely cited with recent references (2019-2022).

Thanks for your suggestion! We replaced some of the obsolete references, but because there are fewer studies for SSIII in the 2019 to 2022 timeframe, some of the obsolete references could not be replaced and had to be retained.

 

8. Figure 4. Transcriptional level regulation of SSIII (Line no. 340): this figure need to be revised with details; how various transcription factors can regulate SSIII. Accordingly detailed legends must be given.’ ‘Even tabular content is not supplemented in any of the section.

Thanks for your suggestion! We have replaced the previous figure with a table that provides a detailed description of the basic information of each transcription factor and the specific ways in which it regulates SSIII. At the same time, we have also created three supplementary tables for the Figures. The purpose is to present the current research progress of SSIII more vividly to the readers in the form of Figures.

(P10, line 337)

Table 1. Information, expression characteristics and regulation of transcription factors regulating SSIII

Transcription factors’ name	Amino Acid	Characteristics of expression	Interaction with SSIII	Reference
ZmEREB156	233aa	Expressed in the roots, stems, leaves and endosperm, and the expression is higher in the endosperm and leaves. Expression is regulated by sucrose or abscisic acid (ABA), and may be synergistically regulated by sucrose and ABA.	Binds directly to the PDull1 and promotes the expression of SSIII.	[69, 70]
ZmEREB192	329aa	Expresses highly in different periods of post-pollinated maize endosperm. Expression is upregulated by sucrose, downregulated by ABA and significantly upregulated by sucrose and ABA.	Binds to the PDull1 weakly and suppress the activity of PDull1.	[71]
ZmEREB25	292aa	Expresses highly in different periods of post-pollinated maize endosperm. Expression is upregulated by sucrose and ABA, significantly upregulated by sucrose and ABA.	Binds directly to the PDull1 and promotes the activity of PDull1.	[71]
ZmEREB26	428aa	Expresses highly in different periods of post-pollinated maize endosperm.	Promotes the activity of PDull1.	[72]
ZmEREB94	283aa	Expressed in the stem, seed, endosperm, and embryo, and the expression is higher in the embryo and endosperm. Expression is upregulated by sucrose and ABA.	Correlate with the expression of SSIII	[73]
ZmbZIP91	563aa	Expresses highly in maize endosperm, strongly associated with the expression of SS genes.	Binds directly to the ACTCAT element of PDull1.	[74]
ZmbZIP22	183aa	Expresses at a relatively high level in endosperm, and the level of expression increases gradually after pollination.	Binds directly to the ACGT element of PDull1.	[75]
Opaque2	405aa	-	Binds directly to the O2 box (ACGT element) of PDull1 and exerted strong activation on its transcription	[76]
ZmWRKY82.2	613aa	Expresses highly in the embryo and different periods of post-pollinated maize endosperm. Expression is upregulated by sucrose, downregulated by ABA and significantly upregulated by sucrose and ABA.	Binds directly to the PDull1.	[77]
ZmPLATZ2	309aa	The expression is highest at 12 DAP and decreases rapidly post 12 DAP. Expression is upregulated by glucose, downregulated by sucrose and ABA.	Promotes the activity of PDull1.	[78]
ZmNAC126	322aa	Expresses highly in the endosperm.	Promotes the activity of PDull1.	[79]
ZmMYB115	517aa	-	Significantly suppress the activity of PDull1.	[80]
ZmGRAS20	508aa	Expresses highly in the endosperm.	Promotes the activity of PDull1.	[72]

 

9. Numbering of section is also need to be checked. I dont know what is the significance of section 8.

In the authors' opinion, the significance of our setting up Section 8 is to show the research method and data source of this paper, and to provide ideas for similar studies to follow. And a similar section setting has been found in previous review papers.

 

10. Further, authors view points are missing from the manuscript. Author must add their view point at end of each section/subsection.

Thanks for your carefully checking! We apologize for this and have made changes and additions.

(P3, 94-97)

Therefore, it can be suggested that the role of SSIII is to form highly ordered amylopectin. The mutants lacking in SSIII would affect the synthesis of amylopectin, reduce the synthetic amount of starch, change the structure, affect the grain weight, and increase the ratio of amylose in starch [33, 34].

 

(P4, 146-149)

In the CBM structural domains β-pleated sheet are found dominant; while in the GT structural domain more α-helixes are recognized (Figure 4). Studies are required to demonstrate the relationship between the distribution of the unique tertiary structure and the function of the structural domain. The exact relationship between the tertiary structure of the peptide chain and the structural domain of SSs needs to be addressed.

 

(P4, 150-155)

Fewer studies have demonstrated the chemical properties of SSIII. It has been shown that the maize SSIII perform the activity with maximum rate at 37℃, whereas it maintains 60% of the maximum activity at 23℃ and the activity will decrease by more than 50% at 42℃ [36]. Similar pattern of activity was observed using the recombinant maize SSIII [43]. Exploring the chemical conditions under which SSIII functions optimally can help in genetic breeding research to to identify the key position of SSIII in maize starch synthesis.

 

(P9, 293-296)

Therefore, it can be suggested that the phosphorylation of starch synthase and the interaction between enzymes could play an important role in regulating starch synthesis and determining the modification of SSIII via phosphorylation and key residues for the phosphorylation could be interesting to be known.

 

 

11. Discussion part need further improvement.’ ‘Please improve the conclusion as per the exact findings of the review article. Current form of conclusion is very crude and need improvement.

Thanks for your suggestion! We have carefully revised the Concluding Remarks and Future Perspectives section to remove unnecessary parts, more accurately summarize the current research progress, and state the authors' outlook on the future direction of SSIII research.

(P12, line 382)

The biosynthesis of starch is a highly complex metabolic process that requires the synergistic action of multiple enzymes. Studies targeting its synthetic pathways have been a long-standing research hot spot, while there are still ongoing controversies and corrections [87]. Existing studies have shown that SSIII acts as the core and regulatory enzyme of maize starch synthase with several important functions including synthesizing highly ordered branched starch, influencing the structure of branched starch and catalyzing the formation of longer sugar chains.

Regarding its physicochemical properties SSIII found to be consisted of a specific transport peptide region. The structure is characterized with a Dull1-specific N-terminal region, a central region (homologous to other class III starch synthesis), and a C-terminal region containing a catalytic structural domain for a total of four functional regions. Several other structural domains including CC, GT5, GT1 and CBM25 have been identified in SSIII, however no direct studies have been conducted to the date to demostrate the functional link of the structural domains with the function of SSIII in starch synthesis. Despite the variations in the sequences the predicted tirtiary structures showed the consistency in the spatial distribution of the structural domains in each of the mentioned isoforms which have not be characterized yet. In terms of its optimal working conditions, only a preliminary study described the effect of temperature on SSIII is available. Considering the central position of SSIII in starch synthesis, further investigation of the optimal physiological conditions of SSIII could be significant for adjusting cultivation practices in maize.

The expression of SSIII found maximum during 10-30 DAP stages and expression of SSIIIa is found dominant in the endosperm. The expression of the starch biosynthetic enzyme significantly affects the starch content of the seeds in these stages suggesting SSIII could also effect on starch yield. The expression results can be utilized to the breeding work of transgenic maize varieties.

In relation to regulation of metabolic pathways several studies have shown that SSIII can be a "regulator" of other key enzymes for starch synthesis including SBE, other SS, and GBSS which act directly or indirectly to participating enzyme. Several reports suggested that SSIII is involved in both starch biosynthesis and cellular glucan metabolism. It is reported to perform several biological activities including glycogen (starch) synthesis activity, starch-binding activity and transferase activity for transferring hexose molecules. SSIII in multiple enzyme complexes with SBEs, SSs, ISA, PUL, PHO and other key enzymes for starch synthesis have been reported in maize and rice crops. However, the pathway of enzyme complex formation and the specific catalytic function of each part of the enzyme complex have not been characterized yet. Furthermore, several enzymes in a complex reported to be modified via phosphorylation and the exact fate of phosphorylation needed further clarifications. phosphorylation starch synthases are thought to be a common phenomenon [53] it can be speculated that SSIII can also modify via phosphorylation, since there is no direct evidence for the phosphorylation have been reported so far, further studies are needed.

Several key elements regulating starch synthesis have been identified in the promoter region of SSIII, however experimental verifications are missing. Further studies are needed to demonstrate the functional importance of the identified elements. Transgenic experiments on PDull1 have shown that PDull1 act as potential tool to improve crop yield. Transcription factors have been identified to regulate the starch synthesis process in plants It can be speculated transcription factors regulating SSII could be influenced by several signaling molecules including sucrose, ABA, miRNAs and set of several other interacting molecules. Considering the important regulatory role of transcription factors we need detection and identification of transcription factors, signaling molecules and miRNAs in future studies which could help to determine the underlying pathways of signaling molecules and miRNAs for the regulation of SSIII function by transcription factors.

 

12. It is hard to establish the link between the various sections.

Thanks for your suggestion! We have adjusted the sections settings and added linking statements between sections to help readers better establish the relationships between sections.

 

13. I am not recommending the review article for publication in “Agronomy”.

We chose the “Agronomy” to provide the readers of “Agronomy “with the latest news on SSIII research progress, and we have seen similar articles published in the “Agronomy”.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Authors have improved the manuscript and efforts are visible.

Manuscript may be accepted in current format. 

Author Response

Authors have improved the manuscript and efforts are visible. Manuscript may be accepted in current format.’

Response; Thank you for reviewing the manuscript again and for your kind comments about it.

Author Response File: Author Response.pdf