Overexpression of HaASR1 from a Desert Shrub, Haloxylon ammodendron, Improved Salt Tolerance of Arabidopsis thaliana

Round 1

Reviewer 1 Report

This manuscript reported the improved salt tolerance in Arabidopsis caused by the overexpression of HaASR1 gene. There was considerable amount of physiological data collected and analyzed to show that the overexpression of HaASR1 had multiple positive effects on salt tolerance. Nevertheless, I do have several questions and concerns. My apologies if I misunderstood or made a mistake.

1. I would be more interested to know more about ASR family genes, such as what essentially are they, how big is this family across plant kingdom, and what are their potential protein functions? These were barely talked about in the text.

2. I believe the MS would benefit more if the author can provide some molecular clues that may be helpful to uncover the mechanisms underline the physiological indices. Molecular evidence, such as the expression of the marker genes involved in the biosynthesis of chlorophyll, proline, and betaine, would be very helpful for supporting the physiological data collected, but there was little molecular data in this MS.

3. There was too much introductory information in the discussion section, whereas a very limited amount of interpretation for the data collected in this study. I think some of the introductory part could be placed into the introduction section and more discussion on the results could be focused on the conclusion drawn from the results as well as insights into the specific mechanism of how ASR functions in plant stress response.

4. Letter a-e corresponding to statistical analysis in all figures need to be annotated.

5. Some places may seem misleading. For example, in result 3.4, “Overexpression of HaASR1 significantly increased chlorophyll a content, Chlorophyll b content and leaf area of transgenic lines under 200 mM NaCl treatment.” I think it would be more precise to describe it as “compared to the WT, the overexpression of HaASR1 significantly prevented the reduction of chlorophyll content and leaf surface area under 200 mM NaCl treatment.”

 

 

Author Response

Reviewer 1:

This manuscript reported the improved salt tolerance in Arabidopsis caused by the overexpression of HaASR1 gene. There was considerable amount of physiological data collected and analyzed to show that the overexpression of HaASR1 had multiple positive effects on salt tolerance. Nevertheless, I do have several questions and concerns. My apologies if I misunderstood or made a mistake.

1. I would be more interested to know more about ASR family genes, such as what essentially are they, how big is this family across plant kingdom, and what are their potential protein functions? These were barely talked about in the text.

Response: Thank you very much for your comments. In 1993, Iusem differentially hybridized cDNA libraries of tomato ripe fruits under drought stress and identified a highly expressed cDNA clone [1], which was subsequently demonstrated to be regulated by ABA [2]. This gene was named abscisic acid, stress, ripening (ASR), ASR expression was induced by water deficit and ABA and it was highly expressed in mature fruits. Tomato ASR1 protein, with a molecular weight of 1.3×10⁴, is rich in alanine, lysine, histidine and glutamic acid and located in the nucleus [1]. Subsequently, ASR were found in a variety of plants, including potato, grape, corn, strawberry, rice, lily, peanut, banana, etc., and the number of ASR varied greatly among different species [3]. ASR has one intron and two exons about 200 bp in length [4], but the length of the first exon of ASR found in corn, sorghum, barley and rice was about 500-600 bp. Taking maize as an example, the first exon of ZmASR3 is 595 bp, while that of other ZmASR is about 200 bp, and the two exons of ZmASR7 are merged together [5], so the structure of ASR in different plants is quite different.

ASR protein is a small molecule and highly hydrophilic protein, containing a large number of polar amino acids such as alanine, lysine, histidine and glutamic acid. It is extremely resistant to high temperature and exists in cells in a highly disordered state. LEA is also a small molecule, highly hydrophilic and heat stable protein. According to sequence similarity and special structure, Ingram divided LEA proteins into six broad categories [6], LEA and ASR protein sequence homology is low, but they are the physical and chemical properties similar to that of induced by water stress, and accumulated in seeds in the process of embryonic development height, so ASR is classed as LEA protein family's seventh largest [7].

ASR proteins can be found in cytoplasm, nucleus and chloroplast. ASR proteins in cytoplasm can be activated by stress signals, and some of them cooperate with other osmotic regulators in the form of monomers to protect stress response proteins. In the nucleus, activated ASR protein acts as a transcription factor to activate the expression of downstream stress response genes, at which time it participates in plant cells' response to environmental signals in the form of dimer. ASR can enhance plant drought resistance, salt tolerance, cold resistance, and regulate fruit development and ripening [8-10].

The relevant content has been added in the article.

Reference

1. Iusem, N.D., Bartholomew, D.M., Hitz, W.D., Scolnik, P.A. Tomato (Lycopersicon esculentum) transcript induced by water deficit and ripening. Plant Physiology, 1993, 102: 1353. https://doi.org/10.1104/pp.102.4.1353.
2. Rossi, M., Iusem, N.D. Tomato (Lycopersicon esculentum) genomic clone homologous to a gene encoding an abscisic acid-induced protein. Plant physiology, 1994, 104: 1073. https://doi.org/10.1104/pp.104.3.1073.
3. González, R.M., Iusem, N.D. Twenty years of research on ASR (ABA-stress-ripening) genes and proteins. Planta, 2014, 239: 941-949. https://doi.org/10.1007/s00425-014-2039-9.
4. Henry, I.M., Carpentier, S.C., Pampurova, S., Van Hoylandt, A., Panis, B., Swennen, R., Remy, S. Structure and regulation of the ASR gene family in banana. Planta, 2011, 234: 785-798. https://doi.org/10.1007/s00425-011-1421-0.
5. Virlouvet, L., Jacquemot, M.P., Gerentes, D., Corti, H., Bouton, S., Gilard, F., Valot, B., Trouverie, J., Tcherkez, G., Falque, M., Damerval, C., Rogowsky, P., Perez, P., Noctor, G., Zivy, M., Coursol, S. The ZmASR1 protein influences branched-chain amino acid biosynthesis and maintains kernel yield in maize under water-limited conditions. Plant physiology. 2011, 157: 917-36. https://doi.org/10.1104/pp.111.176818.
6. Ingram, J., Bartels, D. The molecular basis of dehydration tolerance in plants. Annual review of plant biology, 1996, 47: 377-403. https://doi.org/10.1146/annurev.arplant.47.1.377.
7. Battaglia, M., Olvera-Carrillo, Y., Garciarrubio, A., Campos, F., Covarrubias, A.A. The enigmatic LEA proteins and other hydrophilins. Plant physiology, 2008, 148: 6-24. https://doi.org/10.1104/pp.108.120725.
8. Dai, J.R., Liu, B., Feng, D.R., Liu, H.Y., He, Y.M., Qi, K.B., Wang, H.B., Wang, J.F. MpAsr encodes an intrinsically unstructured protein and enhances osmotic tolerance in transgenic Arabidopsis. Plant Cell Report. 2011, 30: 1219-30. https://doi.org/10.1007/s00299-011-1030-1.
9. Hu, W., Huang, C., Deng, X., Zhou, S., Chen, L., Li, Y., Wang, C., Ma, Z., Yuan, Q., Wang, Y., Cai, R., Liang, X., Yang, G., He, G. TaASR1, a transcription factor gene in wheat, confers drought stress tolerance in transgenic tobacco. Plant Cell Environment. 2013, 36: 1449-64. https://doi.org/10.1111/pce.12074.
10. Pérez-Díaz, J., Wu, T.M., Pérez-Díaz, R., Ruíz-Lara, S., Hong, C.Y., Casaretto, J.A. Organ- and stress-specific expression of the ASR genes in rice. Plant Cell Report. 2014, 33: 61-73. https://doi.org/10.1007/s00299-013-1512-4.
11. I believe the MS would benefit more if the author can provide some molecular clues that may be helpful to uncover the mechanisms underline the physiological indices. Molecular evidence, such as the expression of the marker genes involved in the biosynthesis of chlorophyll, proline, and betaine, would be very helpful for supporting the physiological data collected, but there was little molecular data in this MS.

Response: Thank you very much for your comments. ASR gene can improve plant salt tolerance. Some studies indicate that an ASR gene (GbAsr) has been cloned from Ginkgo biloba, and its expression in roots, stems and leaves can be induced by NaCl, mannitol and ABA [1]. Arabidopsis thaliana lines overexpressing LLA23 showed higher salt tolerance, and the germination rate and dry matter accumulation of transgenic lines were significantly higher than those of the wild type [2]. MpASR of Musa nana can respond to sodium chloride induction. Under high salt stress, Arabidopsis thaliana lines overexpressing MpASR had higher germination rate, longer root system, higher soluble sugar content and less cell damage than the wild type [3]. Our laboratory studies on HaASR of Haloxylon ammodendron focused on cloning HaASR1 and HaASR2 and verification of gene function, and explored the tolerance of transgenic Arabidopsis thaliana lines to salt stress and drought stress [4, 5]. In this paper, compared with the wild type, overexpression of HaASR1 promoted the growth and development of transgenic Arabidopsis thaliana lines under salt stress, and thus improved the plant salt tolerance. In the following work, our laboratory researchers hope to obtain more data to support our research results.

References

1. Shen, G., Pang, Y., Wu, W., Deng, Z., Liu, X., Lin, J., Zhao, L., Sun, X., Tang, K. Molecular cloning, characterization and expression of a novel ASR gene from Ginkgo biloba. Plant Physiological Biochemistry. 2005, 43: 836-43. https://doi.org/10.1016/j.plaphy.2005.06.010.
2. Yang, C.Y., Chen, Y.C., Jauh, G.Y., Wang, C.S. A Lily ASR protein involves abscisic acid signaling and confers drought and salt resistance in Arabidopsis. Plant Physiol. 2005, 139: 836-46. https://doi.org/10.1104/pp.105.065458.
3. Dai, J.R., Liu, B., Feng, D.R., Liu, H.Y., He, Y.M., Qi, K.B., Wang, H.B., Wang, J.F. MpAsr encodes an intrinsically unstructured protein and enhances osmotic tolerance in transgenic Arabidopsis. Plant Cell Report. 2011, 30: 1219-30. https://doi.org/10.1007/s00299-011-1030-1.
4. Gao, H.J., Lü, X.P., Zhang, L., Qiao, Y., Zhao, Q., Wang, Y.P., Li, M.F., Zhang, J.L. Transcriptomic profiling and physiological analysis of Haloxylon ammodendron in response to osmotic stress. International Journal of Molecular Sciences, 2017, 19, 84. https://doi.org/10.3390/ijms19010084.
5. Cao, Y.H., Ren, W., Gao, H.J., Lü, X.P., Zhao, Q., Zhang, H., Rensing, C., Zhang, J.L. HaASR2 from Haloxylon ammodendron confers drought and salt tolerance in plants. Plant Science, 2023, 328, 111572. https://doi.org/10.1016/j.plantsci.2022.111572.
6. There was too much introductory information in the discussion section, whereas a very limited amount of interpretation for the data collected in this study. I think some of the introductory part could be placed into the introduction section and more discussion on the results could be focused on the conclusion drawn from the results as well as insights into the specific mechanism of how ASR functions in plant stress response.

Response: Thank you very much for your comments. We have revised the introduction and discussion part of the paper. Introductory information is added in the introduction, and the experimental results are described more in the discussion. We include references related to the effects of other genes on plant growth and development in the discussion section, because there are few studies on the effects of ASR on plant growth and development under salt stress. Compared with the results of our research on ASR, the introduction of other salt-tolerant genes has the function of promoting plant growth and development. This indicates that the direction of our research on ASR gene is correct and meaningful.

4. Letter a-e corresponding to statistical analysis in all figures need to be annotated.

Response: Thank you very much for your comments. We have reviewed the statistical analysis in all the figures and annotated them.

5. Some places may seem misleading. For example, in result 3.4, “Overexpression of HaASR1 significantly increased chlorophyll a content, Chlorophyll b content and leaf area of transgenic lines under 200 mM NaCl treatment.” I think it would be more precise to describe it as “compared to the WT, the overexpression of HaASR1 significantly prevented the reduction of chlorophyll content and leaf surface area under 200 mM NaCl treatment.”

Response: Thank you very much for your comments. We have checked the description and corrected it.

Reviewer 2 Report

I am a bit concerned with the results presented by the authors in the manuscript "Overexpression of HaASR1 from a desert plant, Haloxylon ammodendron, improved salt tolerance of Arabidopsis thaliana" . Recently these authors published  a paper in Pant Science entitled "HaASR2 from Haloxylon ammodendron confers drought and salt tolerance in plants"  (https://doi.org/10.1016/j.plantsci.2022.111572). 

It is striking that the results plotted for shoot FW and DW are exactly the same using one gene or the other, even the statistical differences between OE1-O3 lines of each gene are exactly the same. 

I want to know which is the difference between HaASR1 (GenBank: MN602038.1) and HaASR2 gene (GenBank accession number: OL908904). I searched both accessions in Genbank and the second one retrieved no results. The authors must demonstrate that these genes differ, they must compare the effect of the OE of both genes and comment on this.

Author Response

Reviewer 2:

I am a bit concerned with the results presented by the authors in the manuscript "Overexpression of HaASR1 from a desert plant, Haloxylon ammodendron, improved salt tolerance of Arabidopsis thaliana". Recently these authors published a paper in Pant Science entitled "HaASR2 from Haloxylon ammodendron confers drought and salt tolerance in plants" (https://doi.org/10.1016/j.plantsci.2022.111572).

It is striking that the results plotted for shoot FW and DW are exactly the same using one gene or the other, even the statistical differences between OE1-O3 lines of each gene are exactly the same.

I want to know which is the difference between HaASR1 (GenBank: MN602038.1) and HaASR2 gene (GenBank accession number: OL908904). I searched both accessions in Genbank and the second one retrieved no results. The authors must demonstrate that these genes differ, they must compare the effect of the OE of both genes and comment on this.

Response: Thank you very much for your comments. ASR (abscisic acid, stress, ripening induced) gene is a class of genes discovered from plants in recent years. Under different environmental stress and fruit ripening stage, the expression of most ASR genes is up-regulated [1-4]. ASR genes mostly exist in plants in the form of gene family, and the number of ASR gene family members varies in different species. For example, there are 9 ASR gene members in maize (Zea mays), only one ASR gene exists in grape, and 5 ASR gene members have been cloned in tomato. At least four members of the ASR gene exist in Musa [5-8]. In the same species, different members of the ASR family have different expression patterns and functions. Tomato ASR1 protein has similar molecular chaperone activity [9], and ASR1 protein is sensitive to water stress in leaves [10]. Tomato ASR2 protein may be involved in carbohydrate metabolism in phloem companion cells [11].

ASR genes exist in most seed plants in family form. In the course of evolution, ASR genes have evolved from common ancestor genes through repetition and differentiation, and they have the same or related functions. The different number of ASR gene family members in different species may be related to the different functions of ASR gene in different species. The evolution of ASR gene families is mainly through gene tandem duplication caused by chromosome unequal exchange, and frequent chromosome unequal exchange leads to coevolution of family members [12]. Tandem duplication may also lead to different evolutionary pathways among ASR gene family members, ultimately contributing to the formation of specific adaptations in plants [13].

The expression of ASR gene family members showed diversity. Under normal culture conditions, ASR2 gene was expressed in phloem companion cells of tomato leaves. In addition to companion cells, a small number of ASR2 gene transcripts were detected in mesophyll cells under water stress. Unlike the ASR2 gene, the ASR1 gene was almost not expressed in vascular tissue either in normal culture or under water stress [11].

Our laboratory has cloned HaASR1 and HaASR2 from desert plant Haloxylon ammodendron, and verified the function of these genes. The results showed that overexpression of HaASR1 and HaASR2 could improve the salt tolerance and drought tolerance of transgenic Arabidopsis thaliana lines compared with WT. For details of the study, reviewer can refer to Gao et al, 2020 and Cao et al, 2023 [14, 15]. In the future, researchers in our laboratory will carry out further research experiments to reveal the functional differences of different ASR gene families.

Reference

1. Kawasaki, S., Borchert, C., Deyholos, M., Wang, H., Brazille, S., Kawai, K., Galbraith, D., Bohnert, H.J. Gene expression profiles during the initial phase of salt stress in rice. Plant Cell. 2001, 13: 889-905. https://doi.org/10.1105/tpc.13.4.889.
2. Bovy, A., de Vos, R., Kemper, M., Schijlen, E., Almenar Pertejo, M., Muir, S., Collins, G., Robinson, S., Verhoeyen, M., Hughes, S., Santos-Buelga, C., van Tunen, A. High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1. Plant Cell. 2002, 14: 2509-26. https://doi.org/10.1105/tpc.004218.
3. Jha, B., Agarwal, P.K., Reddy, P.S., Lal, S., Sopory, S.K., Reddy, M.K. Identification of salt-induced genes from Salicornia brachiata, an extreme halophyte through expressed sequence tags analysis. Genes Genet Syst. 2009, 84: 111-20. https://doi.org/10.1266/ggs.84.111.
4. Chen, J.Y., Liu, D.J., Jiang, Y.M., Zhao, M.L., Shan, W., Kuang, J.F., Lu, W.J. Molecular characterization of a strawberry FaASR gene in relation to fruit ripening. PLoS One. 2011, 6: e24649. https://doi.org/10.1371/journal.pone.0024649.
5. Virlouvet, L., Jacquemot, M.P., Gerentes, D., Corti, H., Bouton, S., Gilard, F., Valot, B., Trouverie, J., Tcherkez, G., Falque, M., Damerval, C., Rogowsky, P., Perez, P., Noctor, G., Zivy, M., Coursol, S. The ZmASR1 protein influences branched-chain amino acid biosynthesis and maintains kernel yield in maize under water-limited conditions. Plant Physiological. 2011, 157: 917-36. https://doi.org/10.1104/pp.111.176818.
6. Cakir, B., Agasse, A., Gaillard, C., Saumonneau, A., Delrot, S., Atanassova, R. A grape ASR protein involved in sugar and abscisic acid signaling. Plant Cell. 2003, 15: 2165-80. https://doi.org/10.1105/tpc.013854.
7. Fischer, I., Camus-Kulandaivelu, L., Allal, F., Stephan, W. Adaptation to drought in two wild tomato species: the evolution of the ASR gene family. New Phytol. 2011, 190: 1032-1044. https://doi.org/10.1111/j.1469-8137.2011.03648.x.
8. Henry, I.M., Carpentier, S.C., Pampurova, S., Van Hoylandt, A., Panis, B., Swennen, R., Remy, S. Structure and regulation of the ASR gene family in banana. Planta. 2011, 234: 785-98. https://doi.org/10.1007/s00425-011-1421-0.
9. Konrad, Z., Bar-Zvi, D. Synergism between the chaperone-like activity of the stress regulated ASR1 protein and the osmolyte glycine-betaine. Planta. 2008, 227: 1213-9. https://doi.org/10.1007/s00425-008-0693-5.
10. Maskin, L., Gudesblat, G.E., Moreno, J.E., Carrari, F.O., Frankel, N., Sambade, A., Iusem, N.D. Differential expression of the members of the ASR gene family in tomato (Lycopersicon esculentum). Plant Science, 2001, 161, 739-746. https://doi.org/10.1016/S0168-9452(01)00464-2.
11. Maskin, L., Maldonado, S., Iusem, N.D. Tomato leaf spatial expression of stress-induced ASR genes. Mol Biol Rep. 2008, 35: 501-5. https://doi.org/10.1007/s11033-007-9114-2.
12. Frankel, N., Carrari, F., Hasson, E., Iusem, N.D. Evolutionary history of the ASR gene family. Gene. 2006, 378: 74-83. https://doi.org/10.1016/j.gene.2006.05.010.
13. Fischer, I., Camus-Kulandaivelu, L., Allal, F., Stephan, W. Adaptation to drought in two wild tomato species: the evolution of the ASR gene family. New Phytol. 2011, 190: 1032-1044. https://doi.org/10.1111/j.1469-8137.2011.03648.x.
14. Gao, H.J., Lü, X.P., Zhang, L., Qiao, Y., Zhao, Q., Wang, Y.P., Li, M.F., Zhang, J.L. Transcriptomic profiling and physiological analysis of Haloxylon ammodendron in response to osmotic stress. International Journal of Molecular Sciences, 2017, 19, 84. https://doi.org/10.3390/ijms19010084.
15. Cao, Y.H., Ren, W., Gao, H.J., Lü, X.P., Zhao, Q., Zhang, H., Rensing, C., Zhang, J.L. HaASR2 from Haloxylon ammodendron confers drought and salt tolerance in plants. Plant Science, 2023, 328, 111572. https://doi.org/10.1016/j.plantsci.2022.111572.

Reviewer 3 Report

The manuscript describes the morphological and physiological assessment on Arabidopsis plants overexpressing HaASR1 gene from Haloxylon ammodendron, a desert xero-halophyte species, under salinity stress. This is an interesting topic, however, the manuscript was not well-written. The introduction is lack of references to support statements, the materials and methods section did not provide sufficient details. The discussion did not provide relevant points and more importantly, one of the parameters that was reported association with expression of ASR gene in other plants under salinity stress– the low Na+ and normal K+ was not presented in this research. Even though in the discussion the author did mention about this point. Overall, the manuscript needs a major revision on both content and English. Please see the attachment for more details.

Comments for author File: Comments.pdf

Author Response

Reviewer 3:

The introduction is lack of references to support statements, the materials and methods section did not provide sufficient details. The discussion did not provide relevant points and more importantly, one of the parameters that was reported association with expression of ASR gene in other plants under salinity stress- the low Na⁺ and normal K⁺ was not presented in this research. Even though in the discussion the author did mention about this point. Overall, the manuscript needs a major revision on both content and English. Please see the attachment for more details.

Response: Thank you very much for your comments. We have added references to the introduction and complement the details in the material approach. We include references related to the effects of other genes on plant growth and development in the discussion section, because there are few studies on the effects of ASR on plant growth and development under salt stress. Compared with the results of our research on ASR, the introduction of other salt-tolerant genes has the function of promoting plant growth and development. This indicates that the direction of our research on ASR gene is correct and meaningful.

ASR can improve plant salt tolerance. Under high salt stress, ASR can reduce the intracellular Na⁺ content, slow down cell damage and maintain osmotic balance. Salicornia brachiata SbASR-1 can improve plant tolerance to high salt stress and impaired fruit development. Under high salt stress, tobacco overexpressing SbASR-1 gene had higher germination rate and lower Na⁺ content [1]. Our laboratory studies on HaASR of Haloxylon ammodendron focused on cloning HaASR1 and HaASR2 from Haloxylon ammodendron and verification of gene function, and explored the tolerance of transgenic Arabidopsis thaliana lines to salt stress and drought stress [2, 3]. In this paper, compared with the wild type, the overexpression of HaASR1 promoted the growth and development of transgenic Arabidopsis thaliana lines under salt stress, and thus improved the plant salt tolerance. In the following work, our laboratory hopes to obtain more data to support our research results.

References

1. Jha, B., Lal, S., Tiwari, V., Yadav, S.K., Agarwal, P.K. The SbASR-1 gene cloned from an extreme halophyte Salicornia brachiata enhances salt tolerance in transgenic tobacco. Mar Biotechnol (NY). 2012, 14: 782-92. https://doi.org/10.1007/s10126-012-9442-7.
2. Gao, H.J., Lü, X.P., Zhang, L., Qiao, Y., Zhao, Q., Wang, Y.P., Li, M.F., Zhang, J.L. Transcriptomic profiling and physiological analysis of Haloxylon ammodendron in response to osmotic stress. International Journal of Molecular Sciences, 2017, 19, 84. https://doi.org/10.3390/ijms19010084.
3. Cao, Y.H., Ren, W., Gao, H.J., Lü, X.P., Zhao, Q., Zhang, H., Rensing, C., Zhang, J.L. HaASR2 from Haloxylon ammodendron confers drought and salt tolerance in plants. Plant Science, 2023, 328, 111572. https://doi.org/10.1016/j.plantsci.2022.111572.

Round 2

Reviewer 1 Report

Most of the concerns and questions have been addressed adequately. However, corroborating molecular data is still strongly recommended and suggested to help level up the paper. 

Author Response

Response: Thank you very much for your comments. In future studies, we will focus on more data to support our existing findings.

Reviewer 2 Report

The authors answered part of my concern in the response to reviewer´s comments letter. However, they have not included any of this in the revised manuscript. They mention that the expression pattern of HaASR1 and HaSR2  differs, that one is associated to the phloem, and that they probably arose by tandem duplication. This they must comment and they must show the differences in sequence of both genes. As I mentioned before, one of the accessions mentioned in the text retrieved no results  so I could not align both sequences. If the 2 proteins are extremely alike it is expected that their OE produces a similar effect. This would confirm the previous results but then there would be no novelty in the present work. They must comment and compare the effect of ASR1-OE with that of ASR2-OE in A. thaliana. 

Author Response

Reviewer 2:

The authors answered part of my concern in the response to reviewer´s comments letter. However, they have not included any of this in the revised manuscript. They mention that the expression pattern of HaASR1 and HaASR2 differs, that one is associated to the phloem, and that they probably arose by tandem duplication. This they must comment and they must show the differences in sequence of both genes. As I mentioned before, one of the accessions mentioned in the text retrieved no results so I could not align both sequences. If the 2 proteins are extremely alike it is expected that their OE produces a similar effect. This would confirm the previous results but then there would be no novelty in the present work. They must comment and compare the effect of ASR1-OE with that of ASR2-OE in A. thaliana. 

Response: Thank you very much for your comments. Our previous results showed that the full length of ASR1 was 382 bp, the length of ORF was 333 bp, the length of 5' untranslated region was 21 bp, the length of 3' untranslated region was 28 bp, encoding 125 amino acids (Fig. 1). Its molecular weight was 14.141 KD and isoelectric point is 6.23. It is rich in alanine (Ala), glutamic acid (Glu), lysine (Lys), histidine (His) and glycine (Gly), which are 15.2%, 15.2%, 12.8%, 10.4% and 7.2%, respectively. Hydrophilic/hydrophobic analysis revealed that ASR1 was a hydrophilic protein (Fig. 2). InterPro tool was used to analyze the ASR1 protein domain and found that it contained highly conserved ABA/WDS domain (Fig. 3), indicating that the cloned target gene was indeed ASR gene, named HaASR1.

 

Fig. 1 Nucleotide sequence and deduced amino acid sequence of ASR1 in H. ammodendron

 

Fig. 2 Hydrophilicity/hydrophobicity analysis of ASR1 protein

 

Fig. 3 Conserved domains analysis of ASR1 protein

In NCBI, similar sequences to HaASR1 were searched by BLAST. The relationship between HaASR1 and Beta vulgaris (XP_010673239.1), Chenopodium quinoa (XP_021735593.1), Spinacia oleracea (XP_021855316.1), Zea mays (NP_001106235.1), Spinacia oleracea (XP_020106501.1), Solanum chilense (the amino acid sequence homology of CBY05743.1) and Suaeda liaotungensis (AGZ20206.1) was 63%, 67%, 61%, 44%, 50%, 46%, and 25%, respectively (Fig. 4). Phylogenetic analysis showed that HaASR1 was closely related to Beta vulgaris (XP_010673239.1), Chenopodium quinoa (XP_021735593.1) and Spinacia oleracea (XP_021855316.1) in the same family, but was far related to maize, rice, tomato and Alpinia liaoning (Fig. 5). The HaASR1 sequence has uploaded NCBI, number: MN602038.1.

 

Fig. 4 Alignments of HaASR1 and another ASR portion. The sources and GenBank accession numbers: AcASR2 (Ananas comosus, XP_020106501.1), AcASR3 (Ananas comosus, OAY74041.1), BvASR1(Beta vulgaris, XP_010673227.1), BvASR2 (Beta vulgaris, XP_010673239.1), BvASR3 (Beta vulgaris, XP_010680922.1), CqASR3 (Chenopodium quinoa, XP_021735593.1), ScASR1 (Solanum chilense, CBY05880.1), ScASR2 (Solanum chilense, CBY05743.1), SoASR2 (Spinacia oleracea, XP_021855316.1), ZmASR (Zea mays，NP_001106235.1), ZmASR1 (Zea mays，ACG35620.1). ABA-WDS motif was indicated with red box, black underlines indicated the amino acid residues of Glu-rich region, His-rich region, two Ala-rich regions and Lys-rich region

 

Fig. 5 Phylogenetic analysis of HaASR1 protein and other ASR portion. SlASR3 is from Suaeda liaotungensis (AGZ2 0206.1), other amino acid sources are in accordance with Figure 4.

The full length of the ASR2 was 479 bp, the ORF was 324 bp, the 5' untranslated region was 48 bp, the 3' untranslated region was 107 bp, encoding 152 amino acids (Fig. 6), the molecular weight was 14.141 KD, and the isoelectric point was 6.23. It contains more alanine (Ala), glutamic acid (Glu), lysine (Lys) and histidine (His), which are 10.5%, 11.8%, 11.2% and 10.5% respectively. Hydrophilic/hydrophobic analysis showed that ASR2 belonged to hydrophilic proteins (Fig. 7). InterPro tool was used to analyze the ASR2 protein domain, and it was found to contain highly conserved ABA/WDS domain (Fig. 8), indicating that the cloned target gene was indeed ASR gene, named HaASR2.

 

Fig. 6 Nucleotide sequence and deduced amino acid sequence of ASR2 in H.ammodendron.

 

Fig. 7 Hydrophilicity/hydrophobicity analysis of ASR2 protein.

 

Fig. 8 Conserved domains analysis of ASR2 protein.

In NCBI, by searching sequences similar to HaASR2 through BLAST, We found that the HaASR2 sequence of Haloxylon ammodendron was correlated with that of beet (Beta vulgaris, XP_010673227.1), Chenopodium quinoa (XP_021735593.1), Spinacia oleracea (XP_021855316.1), corn (Zea mays, NP_001106235.1), pineapple (Spinacia oleracea, XP_021855316.1), Chile tomato (Solanum chilense, The amino acid sequence homology of CBY05743.1 and liaotungens is Liaoz20206.1 was 71%, 68%, 64%, 44%, 51%, 49%, 26%, respectively (Fig. 9). Phylogenetic analysis showed that HaASR2 was closely related to sugar beet (AGZ20206.1) and quinoa (XP_021735593.1), but was far related to maize, rice, tomato and Alpinia Liaoning (Fig. 10). The NCBI of the HaASR2 sequence has been uploaded. The number is OL908904. But the gene has not been made public.

 

Fig. 9 Alignments of HaASR2 and another ASR portion. The sources and GenBank accession numbers: AcASR2 (Ananas comosus, XP_020106501.1), AcASR3 (Ananas comosus, OAY74041.1), BvASR1 (Beta vulgaris, XP_010673227.1), BvASR2 (Beta vulgaris, quinoa，XP_021735593.1), ScASR1 (Solanum chilense, CBY05880.1), ScASR2 (Solanum chilense, CBY05743.1), SoASR2 (Spinacia oleracea, XP_021855316.1), ZmASR (Zea mays, NP_001106235.1), ZmASR1 (Zea mays, ACG35620.1). ABA-WDS motif was indicated with red box, black underlines indicated the amino acid residues of Glu-rich region, His-rich region, two Ala-rich regions and Lys-rich region.

 

Fig. 10 Phylogenetic analysis of HaASR2 protein and other ASR portion. SlASR3 is from Suaeda liaotungensis (AGZ2 0206.1), other amino acid sources are in accordance with Figure 9.

Tissue specific analysis showed that HaASR1 and HaASR2 were mainly expressed in the roots, and the expression level of HaASR1 was 7.72 times that of the control after 350 mM NaCl treatment for 0.5 h, and 10.1 times that of the control after 350 mM NaCl treatment for 1 h. These results indicate that HaASR1 and HaASR2 play important roles in plant response to salt stress. The sequence homology of HaASR2 and HaASR1 is 80%, showing high similarity. Here, we provide the gene sequences and amino acid sequences of HaASR1 and HaASR2 for the reviewers to understand. In future studies, researchers in our laboratory will obtain more data to support our existing findings.

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