GmSTK12 Participates in Salt Stress Resistance in Soybean

Round 1

Reviewer 1 Report

This paper mainly studied the effect of the serine/threonine protein kinase gene GmSTK12 on soybean salt tolerance. The results showed that the overexpression of GmSTK12 resulted in increased salt tolerance by increasing chlorophyll and proline contents as well as the activities of CAT, SOD and POD and by decreasing the MDA and superoxide anion contents. Moreover, the overexpression of GmSTK12 reduced the Na+ content and Na+/K+ ratio. The study suggested that GmSTK12 is essential for salt tolerance in soybean, and provided a theoretical basis for further study of the function of STKs in soybean. The methods is correct, and the data is detailed. However, several problems should be modified:

1.      In introduction, “the relationship between GmSTK12 activity and salt tolerance in soybean is poorly characterized”, there should be many serine/threonine protein kinase genes in soybean, why does the author only focus on GmSTK12? Is there any hint or clue that GmSTK12 is related to salt stress in the previous study? The author should explain it clearly.

2.      In Figure 1, “overexpression (OE, lines 1, 3, and 6) lines” is not clearly showed in Fig.A, please mark the WT, control and OE strains clearly with the symbol in Fig.A.

3.      What is the efficiency of overexpression of GmSTK12 in three OE strains, respectively? The author should present this result in the paper. It is associated with the tested data in this paper among three OE transgenic soybean.

4.      In result 3.3, why were only the leaves and roots as tested material (no other tissues)?

5.      In result 3.3, Na+ contents in leaves of WT is similar with that in roots of WT under no salt stress, why is Na+ contents in leaves of WT higher than that in roots of WT under salt stress? Under salt stress, the roots should be the first to suffer from salt stress.

6.      In result 3.3, under salt stress (200 mmol/L NaCl), why did the K+ content tend to increase in leaves? Why did the K+ content tend to decrease in roots? And, why did the different trends appear in leaves and roots?

7.      In result 3.3, under salt stress, GmSTK12 can maintain the balance of Na+ and K+, and improve the salt tolerance, the effect of GmSTK12 (in OE transgenic soybean) seems to be better in leaves (E) than roots (F), why?

8.      In result 3.4, why were the leaf, stem and root as tested material (why there is no stem in 3.3)?

9.      In result 3.4, GmSOS1 (A), GmSOS2a (B), and GmSOS2b (C) all showed smaller up-regulated expression in leaf than stem and root, why the effect of GmSTK12 (in OE transgenic soybean) seems to be better in leaves than roots that hinted in result 3.3? There seems to be a contradiction.

So, the authors should answer the above questions and explain them in the paper.

Author Response

Response to Reviewer 1 Comments

Point 1: In introduction, “the relationship between GmSTK12 activity and salt tolerance in soybean is poorly characterized”, there should be many serine/threonine protein kinase genes in soybean, why does the author only focus on GmSTK12? Is there any hint or clue that GmSTK12 is related to salt stress in the previous study? The author should explain it clearly.

Response 1: Previous studies have found that GmSTK12 is a downstream gene regulated by GmWRKY20, and GmWRKY20 is an important transcription factor in response to plant stress. Therefore, we studied whether GmSTK12 can also respond to plant adversity and found that GmSTK12 responds strongly to salt stress.

 Text: Abstract. Our previous study identified a transcription factor (GmWRKY20) involved in plant stress resistance, which can directly regulate the expression of GmSTK12.

 

Point 2: In Figure 1, “overexpression (OE, lines 1, 3, and 6) lines” is not clearly showed in Fig.A, please mark the WT, control and OE strains clearly with the symbol in Fig.A.

Response 2: I have marked the WT, control and OE strains clearly with the symbol in Fig.1A

 Text: 3.1 Fig.1A

Point 3: What is the efficiency of overexpression of GmSTK12 in three OE strains, respectively? The author should present this result in the paper. It is associated with the tested data in this paper among three OE transgenic soybean.

Response 3: We analyzed GmSTK12 gene expression levels of three OE strains by QPCR experiment, and the results were shown as follows. And we have put the results into the manuscript.

 Text: 3.1 
Figure 1．(B) GmSTK12 gene relative expression levels of three OE strains

 Point 4: In result 3.3, why were only the leaves and roots as tested material (no other tissues)?

Response 4: The quantitative measure of the Na+and K+ concentrations in roots and shoots in salinized conditions has often been used as a trait in quantitative trait loci mapping, salt tolerance evaluation, and crop breeding programs^[1]. Therefore, we selected soybean roots and salt-sensitive leaves for research, and also verified whether GmSTK12 is involved in Na+ and K+ transport as a protein kinase. 

 Point 5: In result 3.3, Na+ contents in leaves of WT is similar with that in roots of WT under no salt stress, why is Na+ contents in leaves of WT higher than that in roots of WT under salt stress? Under salt stress, the roots should be the first to suffer from salt stress.

Response 5: Yes, root are the first to suffer salt stress. If the Na+ content in plant roots is too high, Na+ is transported from roots to stems and leaves to alleviate ion toxicity and maintain ion dynamic balance.It was found that there was no significant difference in Na+ in the roots of seven cultivated soybeans under 120 mmol/L^-1 NaCl stress, the Na+ content in different tissues of a soybean variety under different salt concentrations, and found that the concentration of xylem entering the leaves determined its salt tolerance^{[2, 3]}.

 Point 6:In result 3.3, under salt stress (200 mmol/L NaCl), why did the K+ content tend to increase in leaves? Why did the K+ content tend to decrease in roots? And, why did the different trends appear in leaves and roots?

Response 6: Yes, we observe that the K+ has different trends in leaves and roots, which is basically consistent with other studies[4]. And there was no difference in the change of K+ between wild type and transgenic plants. We think that GmSTK12 is not involved in K+ regulation, but the regulation of soybean itself.

 Point 7: In result 3.3, under salt stress, GmSTK12 can maintain the balance of Na+ and K+, and improve the salt tolerance, the effect of GmSTK12 (in OE transgenic soybean) seems to be better in leaves (E) than roots (F), why?

Response 7: Thanks for your good suggestion. Roots are the main defensive barrier against salinity and is the organ that first senses this soil-derived stress, leaves are the most sensitive organs to sense salinity^[5]. The Na+ and K+ in the leaves increased, maintaining a high Na+ / K+, reducing leaf damage and maintaining normal physiological functions of the leaves.

 Point 8:In result 3.4, why were the leaf, stem and root as tested material (why there is no stem in 3.3)?

Response 8: Because we found that GmSTK12 is involved in ion transport between aboveground and underground parts of plants, under salt stress, we believe that overexpression of GmSTK12 will affect the expression levels of other genes in different tissues of plants, so the roots, stems and leaves of soybean were studied separately.

 Point 9:In result 3.4, GmSOS1 (A), GmSOS2a (B), and GmSOS2b (C) all showed smaller up-regulated expression in leaf than stem and root, why the effect of GmSTK12 (in OE transgenic soybean) seems to be better in leaves than roots that hinted in result 3.3? There seems to be a contradiction.

Response 9: We think that the function of GmSTK12 is likely to affect Na+ transport to leaves .This is related to the higher expression of GmSOS1, GmSOS2a, and GmSOS2b in root and stem tissues, maintaining ion balance and transferring less Na+ to leaves. Shoot Na+ accumulation is the key factor in performance decline on NaCl stress^[6].We think that this may suggest that GmSTK12 can affect the transport of Na+ to the leaves, or the excessive Na+ in the leaves is exported downward, the final result is reflected in the leaves.

Reference

[1]    REHMAN H M, CHEN S, ZHANG S, et al. Membrane Proteomic Profiling of Soybean Leaf and Root Tissues Uncovers Salt-Stress-Responsive Membrane Proteins. LID - 10.3390/ijms232113270 [doi] LID - 13270 [J]. 1422-0067 (Electronic)):

[2]   VALENCIA R, CHEN P, ISHIBASHI T, et al. A rapid and effective method for screening salt tolerance in soybean [J]. Crop science, 2008, 48(5): 1773-9.

[3]    DURAND M, LACAN D. Sodium partitioning within the shoot of soybean [J]. Physiologia Plantarum, 1994, 91(1): 65-71.

[4]    ZHANG M, CAO J, ZHANG T, et al. A Putative Plasma Membrane Na(+)/H(+) Antiporter GmSOS1 Is Critical for Salt Stress Tolerance in Glycine max [J]. 1664-462X (Print)):

[5]    MUNNS R, TESTER M. Mechanisms of salinity tolerance [J]. 1543-5008 (Print)):

[6]    COSKUN D, BRITTO DT FAU - JEAN Y-K, JEAN YK FAU - KABIR I, et al. K+ efflux and retention in response to NaCl stress do not predict salt tolerance in contrasting genotypes of rice (Oryza sativa L.) [J]. 1932-6203 (Electronic)):

Author Response File: Author Response.docx

Reviewer 2 Report

The article of  Yang Liu et al.   "The GmSTK12 Gene Participates in Salt Stress Resistance in Soybean” presents analysis of the soybean plants overexpressing GmSTK12 gene. Modern methods were used in the research and a sufficient amount of data was collected. The article corresponds to the profile of "Agronomy" and could be interesting for their readers. However, some small faults should be corrected before publication, especially in the Section “Materials and Methods”.

1) Section 2.3, authors write: “The relative gene expression levels were calculated using the 2^−ΔΔCt method”

- Please add more details about the method or provide a relevant reference.

2) Section 2.4: “The superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities, as well as the contents of hydrogen peroxide (H2O2), proline (PRO), and malondialdehyde (MDA), were measured with commercial Boxbio kits (Beijing Boxbio Science & Technology Co., Beijing, China)”.

- It is necessary to describe the protocols used in more detail. In particular, please indicate which methods are used in the commercial kits and provide relevant references. This is very important as different methods can be used to determine the biochemical parameters mentioned above.

 

Author Response

Response to Reviewer 2 Comments

Point 1: Section 2.3, authors write: “The relative gene expression levels were calculated using the 2^−ΔΔCt method”

- Please add more details about the method or provide a relevant reference.

Response 1: Thanks for your good suggestion. I have provided a relevant reference the 2^−ΔΔCt method^[1].

 Text: 2.3. 2^−∆∆Ct=[(Ct _{gene of interest} – Ct _{internal control}) _{sample A}-(Ct _{gene of interest} – Ct _{internal control}) _{sample B}])

 Point 2: Section 2.4: “The superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities, as well as the contents of hydrogen peroxide (H2O2), proline (PRO), and malondialdehyde (MDA), were measured with commercial Boxbio kits (Beijing Boxbio Science & Technology Co., Beijing, China)”.

- It is necessary to describe the protocols used in more detail. In particular, please indicate which methods are used in the commercial kits and provide relevant references. This is very important as different methods can be used to determine the biochemical parameters mentioned above.

Response 2: Specific experimental methods have been added to the article. The details are as follows.

Text: 2.4. “The superoxide dismutase (SOD, nitro-blue tetrazolium method, AKAO001M), catalase (CAT, ammonium molybdate method, AKAO003-2M), and peroxidase (POD, guaiacol method, AKAO005M) activities, as well as the contents of proline (PRO, acidic ninhydrin method, AKAM003M), and malondialdehyde (MDA, TBA (thiobarbiturate) method, AKFA013M), were measured with commercial Boxbio kits (Beijing Boxbio Science & Technology Co., Beijing, China).”

 Reference

 [1]  SCHMITTGEN T D, LIVAK K J. Analyzing real-time PCR data by the comparative CT method [J]. Nature protocols, 2008, 3(6): 1101-8.

Author Response File: Author Response.docx

Reviewer 3 Report

The work deals about the role of GmSTK12 on soybean salt tolerance.

The results obtained suggest that GmSTK12 is essential for salt stress resistance in soybean and may be used to improve soybean yield and quality.

We think that the manuscript needs to be improved, following suggestions below:

-In the title "GmSTK12 gene" is not appropriate.

- In the abstract the sentence “the results indicate that GmSTK12….for salt tolerance in soybean” is not appropriate, because you can only hypothesize the involvement of GmSTK12 in the complex mechanism of soybean response to salt stress. In the results, discussion and conclusions you should underlined the probable role of GmSTK12 in resistance, or better in salt stress tolerance.

-The introduction is very long, you should describe only the main mechanism of action of serine/threonine protein kinases. 

-I think that figure 1A is not clear.

- In paragraph 3.3 I think that is not GmSTK12 gene to reduce the accumulation of Na⁺ but is the protein.

- About gene expression analysis I think that data are not adequately presented by heat map. Moreover in material and method section you should explain how calculate relative expression using 2^-DDCt.

- In the discussion section you should better explain because no significant difference of K⁺ was observed between treatments and because this may be related to gene expression data about GmSOS1, GmSOS2a and GmSOS2b.

- In paragraph 4.3 is not clear the role of organic solute and inorganic ions, also in relation to the literature. What do you mean “Additionally, overexpression plants…….more proline.

- In the conclusions section you should underline that the overexpression of GmSTK12 probable influence the soybean response to salt stress.

-The English language must be improved.

Author Response

Response to Reviewer 3 Comments

Point 1: In the title "GmSTK12 gene" is not appropriate

Response 1: Thanks for your good suggestion. I have change the GmSTK12 gene to GmSTK12.

 

Text: GmSTK12 Participates in Salt Stress Resistance in Soybean

 

Point 2: In the abstract the sentence “the results indicate that GmSTK12….for salt tolerance in soybean” is not appropriate, because you can only hypothesize the involvement of GmSTK12 in the complex mechanism of soybean response to salt stress. In the results, discussion and conclusions you should underlined the probable role of GmSTK12 in resistance, or better in salt stress tolerance.

Response 2: Thanks for your good suggestion. We improve the accuracy of words.

Text:

Abstract. Taken together, the results indicate that GmSTK12 involved in the mechanism of soybean response to salt stress.

4.2. It likely appears that GmSTK12 enhances the activity of the antioxidant system in soybean, resulting in decreased membrane lipid damage and oxidative stress and increased salt tolerance.

5. Conclusions, Furthermore, overexpression of GmSTK12 likely influences the soybean response to salt stress.

 

Point 3: The introduction is very long, you should describe only the main mechanism of action of serine/threonine protein kinases.

Response 3: We accept your valuable advice,We have revised the introduction.

 

Point 4: I think that figure 1A is not clear.

Response 4: I have marked the WT, control and OE strains clearly with the symbol in Fig.A

Text: 3.1 Fig.1A

 

Point 5: In paragraph 3.3 I think that is not GmSTK12 gene to reduce the accumulation of Na+ but is the protein.

Response 5: Your suggestion is correct, we change the GmSTK12 gene to GmSTK12。

 

Point 6: About gene expression analysis I think that data are not adequately presented by heat map. Moreover in material and method section you should explain how calculate relative expression using 2^−ΔΔCt.

Response 6: The heat map data is drawn according to the relative expression of genes, in order to more intuitively show the expression level of genes in different tissues and different periods. I have provided a relevant reference the 2^−ΔΔCt method^[1].Results were plotted as heat maps using TBtools.

 

Text: 2.3. 2^−∆∆Ct=[(Ct _{gene of interest} – Ct _{internal control}) _{sample A}-(Ct _{gene of interest} – Ct _{internal control}) _{sample B}])

 

Point 7: In the discussion section you should better explain because no significant difference of K+ was observed between treatments and because this may be related to gene expression data about GmSOS1, GmSOS2a and GmSOS2b.

Response 7: Your advice is very important to us, and our presentation here is somewhat unclear. We want to express that the change trend of Na+ in plants may be related to the expression of SOS gene, while the change of K+ is not directly related to the expression of SOS gene. From this study, it can also be seen that there is no significant difference in K+ changes between GmSOS1 mutant and wild type^[2].

 

Point 8: In paragraph 4.3 is not clear the role of organic solute and inorganic ions, also in relation to the literature. What do you mean “Additionally, overexpression plants…….more proline.

Response 8: We further clarified the role of inorganic ions and organic solutes in plant stress resistance. For proline, we want to express that proline is an important osmotic regulator, and GmSTK12 can enhance plant salt tolerance by affecting the accumulation of proline.

 

Text: 4.3 These osmolytes participate in the regulation of osmotic pressure by lowering the osmotic potential in the cytosolic compartment

 

Point 9: In the conclusions section you should underline that the overexpression of GmSTK12 probable influence the soybean response to salt stress.

Response 9: Thanks for your good suggestion.I have underline the question in the conclusions section.

 

Text: 5. Conclusions, Furthermore, overexpression of GmSTK12 likely influences the soybean response to salt stress.

 

Point 10: The English language must be improved..

Response 10: I have my manuscript checked by a native English-speaking colleague.

Reference

[1]    SCHMITTGEN T D, LIVAK K J. Analyzing real-time PCR data by the comparative CT method [J]. Nature protocols, 2008, 3(6): 1101-8.

[2]    ZHANG M, CAO J, ZHANG T, et al. A Putative Plasma Membrane Na(+)/H(+) Antiporter GmSOS1 Is Critical for Salt Stress Tolerance in Glycine max [J]. 1664-462X (Print)):

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The revised manuscript is suitable for the publication.