Analysis of the NAC Gene Family in Salix and the Identification of SpsNAC005 Gene Contributing to Salt and Drought Tolerance
Round 1
Reviewer 1 Report
Dear Editor, Dear Authors,
Thank you very much for the manuscript. The work describes bioinformatic analysis of a few members of the large family of transcription factors NAC from Salix psammophila and characterization of the one protein from this group SpsNAC005 through the means of bioinformatics, gene expression analysis and genetic transformation considering also the salt and drought conditions.
The results showed that transgenic plants are more vigorous under these stresses as was shown by morphological, biochemical and gene expression parameters of transgenic plants. The work overall is quite complete and relevant in the context of changing climate. In my opinion it is publishable, but some corrections must be made before the publication.
In addition to English, some other corrections are needed, so I list my observations below.
Line 2-3. For me something is missing from the title phrase “the identification of SpsNAC005 gene to salt and drought tolerance”. May be better sound “the identification of SpsNAC005 gene contributing/relevant to salt and drought tolerance” or something similar.
Line 12. As the procedure of isolation is not described in the method section it is not quite clear does the 5 genes were indeed isolated from Salix psammophila or only bioinformatically characterized.
Lines 21-23. I would better interchange the results describing enzyme activity with gene expression results in abstract, because in such an order the results are presented in the results section.
Line 31. As this is the first mention in the text, I would like to suggest transcribing abbreviation NAC.
Line 33. Indeed, the first genes identified in NAC family were ATAF1 and ATAF2. Here I present a fragment from the paper Jed A Christianson, Elizabeth S. Dennis, Danny J Llewellyn & Iain W. Wilson, 2010. ATAF NAC transcription factors: Regulators of plant stress signaling. Plant Signaling & Behavior, vol.5: 428-432: “ATAF1 and ATAF2 from Arabidopsis were some of the first described NAC members. The sequences of these genes were first deposited in Genbank in 1993 (Hirst H, University of Vienna) and were named because of their ability to activate the cauliflower mosaic virus 35S promoter in yeast (Arabidopsis thaliana activation factor), although the work was never formally published”.
Line 37. The citation [3] is quite complex for the cited purpose. I‘d better choose a review article which is simpler for common understanding.
Line 37. “The NAC gene family is generally composed” editable style
Lines 37-38. “Substructural domains” I’d change to “Structural subdomains”.
Line 39. The conservatism of structural subdomains is poorly described in [4], the citation [29] would be preferable.
Line 48. Citation 8 is not appropriate here in my opinion, because it describes another protein family (Homeodomain-leucine zipper).
Lines 55-56. “oxidation” I’d change to “oxidative stress”.
Line 59. [13] paper is difficult to find, should be better described in references.
Lines 61-62. Increased by 22-34% and not yield, but seed setting.
Lines 63-72. It is a little pity, that the literature about Salix psammophila is mainly in chinesse language and is hardly to find. Also there are no any explanations throughout all manuscript about the reasons that led to the choice of Populus hopeiensis for genetical transformation. Is this important plant for industry or was chosen as laboratory model?
Lines 77-78. Because there is no description of isolation, remains unclear why namely these 5 genes were chosen for bioinformatic analysis. Some justification or explanation would be appropriate here. Also because of that, there is no description of Salix psammophila plants, from which these genes were isolated.
Lines 79-86. At least earlier we should cite the online resources, which we used in our works. I don’t know, maybe it is not necessary now, but in some online sources such request remains.
Line 79. For https://web.expasy.org/protscale/ the scale used would be appropriate to mention.
Line 90. The online source of the Phytozome database is not mentioned.
Lines 91 and 93. “NAM” should be “NAC”.
Line 92. HMMER software version would be appropriate to specify as well as the source.
Line 92. “NAC gene” to “NAC genes”.
Line 98. MEGA X software version would be appropriate to specify.
Line 103. One fragment I suppose.
Line 110. Test strains I suppose.
Lines 110-114. In this section I lack the description of Salix psammophila plants (source, age, growth conditions), which were used for 5 NAC gene isolation and SpsNAC005 gene expression analysis. The construction of SpsNAC005 gene vector also is not described in methods nor reference specified.
Line 128. Only Primer3 input I found in the online source specified.
Lines 135-136. The primer sequences specified are for reference gene (ubiquitin). The primers for SpsNAC005 gene expression analysis are not mentioned here.
Lines 143-144. The source from which PtrActin primers sequences were obtained or primer design software used would be appropriate to specify.
Lines 181-184. The sources of primer sequences or primer design software used would be appropriate to specify.
Lines 187-193. It is not quite clear in which investigation the “two sample ANOVA” was used and what kind of test it is. Is this two-way ANOVA? The term equal/heteroskedasticity hypothesis is quite unclear for me. I suppose that you tested data for variance equality with F test before t test, as described under the figures, but it is appropriate to mention this also in the statistics section. The complicated terms are “one-way randomized area group test ANOVA” and “ANOVA for one-way randomized block group”. One-way ANOVA for randomized block design maybe appropriate term or something similar. The letter “P” denoting p-value should be uniformed as “P” (capital) or “p” (lowercase) throughout all the manuscript and “P>0.05, not significant” is not necessary to mention at all.
Line 195. The procedure of cloning 5 genes is not described in methods.
Line 197. The procedure of isolation 5 genes is not described in methods.
Line 216. The reference 28 (the same as reference 3) is a little bit too complex. The reference 29 is quite sufficient.
Line 220. “Basic information of SpsNAC005, SpsNAC034, SpsNAC041, SpsNAC042, SpsNAC120 in Salix psammophila.” I’d rewrite as “Basic information of SpsNAC005, SpsNAC034, SpsNAC041, SpsNAC042, SpsNAC120 proteins in Salix psammophila”.
Table 1. The terms are confused. Hydropathicity should be instead of Hydrophobicity and all proteins are hydrophilic (instead of hydropathicity).
A little attention in the manuscript is paid for SpsNAC041 and SpsNAC042, though they differ from other NAC proteins by their structure and predicted localization in the cell. It would be interesting to know more about them.
Line 262. Reference 32 is the same as reference 2.
Line 273. ANAC072 is omitted from description, though it is mentioned in the reference 36.
Line 282. The abbreviation UPR should be transcribed.
Lines 289-290. There exist two possibilities for the difference between Salix and Arabidopsis in this case. The genes may be lost, indeed, in Arabidopsis, but from the other side they can be acquired de novo in Salix.
Line 293. Because Salix and Arabidopsis, both, are dicots, the explanation, that NAC subgroups l, p and q contains proteins only from Arabidopsis, because of differences between monocots and dicots, is insufficient or could be even wrong. Another explanation should be submitted here.
Although 5 isolated proteins were presented in the phylogenetic tree (Fig. 2), but no description of their dependence to various subgroups are presented in the text except SpsNAC005, so, the isolated proteins seems incompletely bioinformatically characterized.
Figure 2. Because of similarity in some colors, it is quite difficult to discriminate subgroups only by color in the figure. It would be very practically to indicate subgroups also by letters in the figure, similarly, as was done in reference 26.
Lines 309-311. The statement “Previous studies have shown that expression of the Arabidopsis ATAF1 gene, which is also in NAC-d（Ⅱ） subfamily, was significantly induced by high salinity and abscisic acid (ABA)” needs citation.
Line 316. The reference 41 is not appropriate here, because environmental stress or leaf senescence were not investigated in the cited work. Also, the reference 37 is not appropriate here, because drought or salinity were not investigated in the presented reference.
Line 346. The difference was not significant between semi-lignified stems, shoots, and also mature stems.
Line 373. Enhance only the height, but not height growth.
Line 376. Statistically analyzed could be only data and leaf areas should be measured before. Style
Figure 6. The letters of subfigures B and C are confused between each other. Also, titles of y-axis “Plant height/cm” and “Leaf area/cm2” better to write “Plant height (cm)” and “Leaf area (cm2)” or “Plant height, cm” and “Leaf area, cm2”.
Lines 405-412. Figure caption should be arranged with one title. It would be proper to mention the number of plants from which the mean was derived. The letter “P” denoting p-value should be uniformed as “P” (capital) or “p” (lowercase). “*0.01 ≤ P < 0.05” and “P>0.05, not significant” is not necessary to mention.
3.7 section. Diameter in this section should be defined as ground diameter
Lines 443-444. Phrase “growth rate” repeats two times.
Lines 443-450. Figure caption should be arranged with one title. It would be proper to mention the number of plants from which the mean was derived. The letter “P” denoting p-value should be uniformed as “P” (capital) or “p” (lowercase). “P>0.05, not significant” is not necessary to mention.
Line 457. All the values of SOD, POD and MDA are expressed as percent, except SOD and this brings some confusion.
Lines 517-522. Figure 8. The term “physiological indicators” must be explained in more detail – which type of indicators – relevant to stress or other? SOD, POD and MDA values sounds not quite right, better SOD activity, POD activity and MDA content or similar. Also, as in earlier figure captions the letter “P” denoting p-value should be uniformed as “P” (capital) or “p” (lowercase). “P>0.05, not significant” is not necessary to mention.
Line 526. Which type of markers? Does it mean tolerance to salinity markers or something else?
Lines 585-591. Figure 9. It is better to mention first salt and after drought in capture as it is in the figure. Also, the letter “P” denoting p-value should be uniformed as “P” (capital) or “p” (lowercase). “P>0.05, not significant” is not necessary to mention as in earlier figure captions. I don’t see any SD in the figure. Does it mean, that experiments results coincided each other quite perfect? The number of experiments is not specified.
Lines 604-606. A little bit confusing sentence “The results of this study were consistent with the phenotype of ANAC032 overexpression plants in Arabidopsis thaliana, and the same phenotype was observed in ANAC032 homologous overexpression plants.” Please, check.
Line 618. ANAC032 is not mentioned in reference 46.
Line 619. In my opinion, mistakenly cited reference 48, because the gene SNAC1 investigated here is not the same as OSNAC045, mentioned in the manuscript.
Line 630. Is this indeed this reference? Because in the reference 53 KCl, CdCl2 and PEG factors are not mentioned.
Line 671. The reference might be inappropriate because the study referenced as 62 investigates MPK4 rather than MPK6.
Line 674. It should be mentioned, that “MPK6 gene was positively correlated with the expression of the SpsNAC005 gene” under stress conditions.
Line 678. Please, recheck the statement as in my opinion, HKT has a high affinity to Na+ and is inhibited by K+.
Line 690. It worth to mention plant species of seven transgenic lines. Also, it is sometimes used term “lines” and sometimes term “strains” (as for viruses and bacteria). It would be better to uniform the term throughout all manuscript. I’d prefer “lines”.
Line 699. There is no evidence in the manuscript, that salt and drought stresses induce the expression of SpsNAC005 gene.
Line 706. Carrier=vector?
Lines 706-707. J.W., X.G. and L.L are not the authors of the manuscript and authors D.W. and G.Z. does not contributed to the manuscript.
Line 753. The reference 13 lacks the title.
Line 765. The reference 17 lacks volume number.
Line 784. The reference 25 lacks the date of last accession.
Line 790. The reference 28 is the same as reference 3.
Line 767. The reference 32 is the same as reference 2.
Line 818. The reference 39 lacks journal title.
Line 824. The list of authors of reference 42 is not complete, in my opinion. Please check.
Line 829. The reference 44 should be cited as dissertation.
Line 842. The reference 49 should contain pages 174-181.
Lines 844-847. The references 50 and 51 must be interchanged.
Lines 852-853. Some online sources give the title of journal of reference 54 as Acta Agronomica Sinica. Please, check.
Line 855. The reference 55 should be cited as dissertation.
Hope my comments will be helpful. Thank you, very much and good luck
The same comments I added in additional file
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript submitted by Yang et al have tried to put important information by using the analytical approach with further gene expression, and a few biochemical inputs of the NAC gene family in Salix. This was followed by the identification of the SpsNAC005 gene response to salt and drought tolerance. My main questions to the authors about the study are as follows:
Firstly, in Fig. 1, Fig. 2, and Fig. 3- the resolution is very poor, and should be changed with a good resolution picture, the text size should be increased in Fig 3.
Did the authors check the protein content of all the OE lines by western blot and correlated it with the transcript levels?
What about the fresh weight and dry weight of OE lines 4, 3, and 1?
How did the authors collect the sample for semi-lignified stems? On what basis do they select such sample regions on trees?
What about the proline content in the OE lines on salt and drought treatment? Authors should check it.
Authors should also check the expression pattern of P5CS1 and PRODH1 genes under salt and drought stress in such lines.
Line 533- change it to the -expression pattern
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
Thank you for your answers to my questions and comments.
