Document Type : Research Paper


1 Department of Plant Breeding and Biotechnology, Agricultural Sciences and Natural Resources University, Sari, Iran

2 Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University, Sari, Iran

3 Crop and Horticultural Science Research Department, Khorasan Razavi Agricultural and Natural Resources Research and Education Center, AREEO, Mashhad, Iran


Salinity is one of the most important limitation factors in development of agricultural products. Cotton has a relative tolerance to salinity; however, salinity reduces its growth during germination and seedling stages. In this research, split-factorial design of time based on randomized complete block design with 3 replications was used. The real-time PCR results for, root, stem, and leaves of 14-day cotton seedlings of tolerant (Sepid) and sensitive (Thermus14) cotton cultivars with salinity levels from 0 to 16 ds.m-1 were analyzed at three time points, namely 0, 7 and 14 days after salinity stress. Selected genes for Real Time PCR reaction in current study were selected using Cytoscape 3.3.0 software. Results showed that the selected genes GhERF2, GhMPK2, GhCIPK6, GbRLK, GhNHX1, GhGST, GhTPS1 and Gh14-3-3 have positively responded to salinity stress and their expression in the root was higher than in stem and leaf. Moreover, the expression of tolerant genotype (Sepid) was higher than the sensitive cultivar (Thermus 14) one, however, a slight increase in sensitive genotypes was observed in a number of genes (GhERF2 and GhGST) 14 days after starting the stress treatment.


Main Subjects

[1]     Ahmad, S., Khan, N., Iqbal, M., Hussain, A. and Hassan, M. 2002. Salt tolerance of cotton (Gossypium hirsutum L.). Asian J Plant Sci, 1:715–719.
[2]     Apse, M.P., Aharon, G.S., Snedden. W.S. and Blumwald. E. 1999. Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Sci; 285:1256–1258.
[3]     Beckers, G.J., Jaskiewicz, M., Liu, Y., Underwood, W.R., He, S.Y., Zhang, S. and Conrath, U., 2009. Mitogen-activated protein kinases 3 and 6 are required for full priming of stress responses in Arabidopsis thaliana. Plant Cell, 21: 944–953
[4]     Blumwald, E., Cragoe, E.J., Poole, R. J. 1985. Na+/H+ antiport in isolated tonoplast vesicles from storage tissue of Beta vulgaris. Plant Physiol, 78, 163-167.
[5]     Borgatti, S.P., Everett, M.G. 2006. A graph-theoretic perspective on centrality. Soc Networks 28: 466–484.
[6]     Champion, A., Hebrard, E., Parra, B., Bournaud, C., Marmey, P., Tranchant, C. and Nicole, M. 2009. Molecular diversity and gene expression of cotton ERF transcription factors reveal that group IXa members are responsive to jasmonate, ethylene and Xanthomonas. Mol. Plant Pathol. 10: 471–485.
[7]     Chan, C. and Lam, H.M. 2014. A putative lambda class glutathione S-transferase enhances plant survival under salinity stress. Plant Cell Physiol, 55, 570–579.
[8]     Chauhan, S., Forsthoefel N., Ran, Y., Quigley, F., Nelson, D. E. and Bohnert, H. J. 2000. Na+/ myo-inositol symporters and Na+/H+-antiport in Mesembryan themumcrystallinum. The Plant J, 24, 511–522.
[9]     Chen, L., Ren, F., Zhou, L., Wang, Q.Q., Zhong, H. and Li, X.B. 2012. The Brassica napus Calcineurin B-Like 1/CBL-interacting protein kinase 6 (CBL1/CIPK6) component is involved in the plant response to abiotic stress and ABA signaling. J Exp Bot, 63(417): 6211–6222.
[10]  Chi, Y., Cheng, Y., Vanitha, J., Kumar, N., Ramamoorthy, R., Ramachandran, S. and Jiang S.Y. 2011. Expansion mechanisms and functional divergence of the glutathiones-transferase family in sorghum and other higher plants. DNA Res, 18, 1–16.
[11]  Cruts, M., Theuns, J. and Broeckhoven, C.V. 2012. Locus-specific mutation databases for neurodegenerative brain diseases. Hum Mutat, 33(9): 1340–1344.
[12]  Dong, Y., Li, C., Zhang, Y., He, Q., Daud, M.K., Chen, J. and Zhu, S. 2016. Glutathione S-Transferase Gene Family in Gossypium raimondii and G.arboreum: Comparative Genomic Study and their Expression under Salt Stress., Front Plant Sci, 7, 139.
[13]  Droillard, M.J., Boudsocq, M., Barbier-Brygoo, H. and Lauriere, C. 2004. Involvement of MPK4 in osmotic stress response pathways in cell suspensions and plantlets of Arabidopsis thaliana: activation by hypoosmolarity and negative role in hyperosmolarity tolerance. FEBS Lett, 574:42–48.
[14]  Ebrahimie, M., Esmaeili, F., Cheraghi, S., Houshmand, F., Shabani, L. and Ebrahimie, E. 2014. Efficient and Simple Production of Insulin-Producing Cells from Embryonal Carcinoma Stem Cells Using Mouse Neonate Pancreas Extract, As a Natural Inducer. PLOS ONE 9(3): e90885.
[15]  Finkelstein, R.R., Gampala, S.S. and Rock, C.D. 2002. Abscisic acid signaling in seeds and seedlings. Plant Cell, 14: S15–S45.
[16]  Finnie, C, Andersen, C.H., Borch, J., Gjetting, S., Christensen, A.B., de Boer A.H., Thordal-Christensen, H. and Collinge, D.B. 2002. Do 14-3-3 proteins and plasma membrane H+-ATPases interact in the barley epidermis in response to the barley powdery mildew fungus. Plant Mol Biol, 49: 137–147.
[17]  Fruzangohar, M., Ebrahimie, E. and Adelson, D.L. 2014. Application of global transcriptome data in gene ontology classification and construction of a gene ontology interaction network. bio Rxiv, 004911.
[18]  Fruzangohar, M., Ebrahimie, E., Ogunniyi, A.D., Mahdi, L.K., Paton, J.C. and Adelson, D.L. 2013. Comparative GO: a web application for comparative gene ontology and gene ontology-based gene selection in bacteria. PLOS ONE 8, e58759.
[19]  Fujimoto, S.Y., Ohta, M., Usui, A., Shinshi, H. and Ohme-Takagi, M. 2000. Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression. Plant Cell, 12:393–404.
[20]  Fukuda, A., Chiba, K., Maeda, M., Nakamura, A., Maeshima M. and Tanaka, Y. 2004. Effect of salt and osmotic stresses on the expression of genes for the vacuolar H+-pyrophosphatase, H+-ATPase subunit A, and Na+/H+ antiporter from barley, J Exp Bot, 55(397): 585-594.
[21]  Galvan-Ampudia, C. S. and Testerink, C. 2011. Salt stress signals shape the plant root. Curr. Opin. Plant Biol, 14: 296–302
[22]  Gao, S.Q., Chen, M., Xia, L.Q., Xiu, H.J., Xu, Z.S., Li, .C., Zhao, C.P., Cheng, X.G. and Ma, Y.Z. 2009. A cotton (Gossypium hirsutum) DRE-binding transcription factor gene, GhDREB, confers enhanced tolerance to drought, high salt, and freezing stresses in transgenic wheat. Plant Cell Rep, 28: 301–311.
[23]  Gu, L., Liu, Y., Zong, X., Liu, L., Li, D.P. and Li, D.Q. 2010. Overexpression of maize mitogen-activated protein kinase gene, ZmSIMK1 in Arabidopsis increases tolerance to salt stress. Mol Biol Rep, 37: 4067–4073.
[24]  Guo, Y.H., Yu, Y.P., Wang, D., Wu, C.A., Yang, G.D., Huang, J.G. and Zheng, C.C. 2009. GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5, New Phytol, 183: 62–75.
[25]  Halfter, U., Ishitani, M. and Zhu, J. 2000. The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3. Proc Natl Acad Sci USA, 97:37-35.
[26]  Hamada, A., Shono, M., Xia, T., Ohta, M., Hayashi, Y., Tanaka, A. and Hayakawa, T. 2001. Isolation and characterization of a Na+/H+ antiporter gene from the halopyte Atriplex gmelini. Plant Molecular Biology, 46: 35-42.
[27]  Hasegawa, P.M., Bressan, R.A., Zhu, J.K. and Bohnert, H.J. 2000. Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol, 51:463–499.
[28]  He, L., Yang, X., Wang, L., Zhu, L., Zhou, T., Deng, J. and Zhang, X. 2013. Molecular cloning and functional characterization of a novel cotton CBL-interacting protein kinase gene (GhCIPK6) reveals its involvement in multiple abiotic stress tolerance intransgenic plants, Biochem Biophys Res Commun, 435: 209–215.
[29]  He, C., Fong, S.H., Yang, D. and Wang, G.L. 1999. BWMK1, a novel MAP kinase induced by fungal infection and mechanical wounding in rice. Mol Plant Microbe Interact, 12:1064–1073
[30]  Hirt, H. 1997. Multiple roles of MAP kinases in plant signal transduction. Trends Plant Sci, 2(1):11–15.
[31]  Hoagland, D.R. and D.I. Arnon. 1950. The water culture method for growing plants without soil. Circular 347, College of agriculture, University of California.
[32]  Hsu, C.W., Juan, H.F. and Huang, H.C. 2008. Characterization of microRNA regulated protein‐protein interaction network. J Proteom, 8:1975–1979.
[33]  Hua, D.P., Wang, C., He, J, Liao, H., Duan, Y., Zhu, Z.Q., Guo, Y., Chen, Z.Z. and Gong, Z.Z. 2012. A plasma membrane receptor kinase, GHR1, mediates abscisic acid-and hydrogen peroxide-regulated stomatal movement in Arabidopsis. Plant Cell, 24:2546–2561.
[34]  Huang, G.Q., Li, W., Zhou, W., Zhang, J.M., Li, D.D., Gong, S.Y. and Li, X.B. 2013. Seven cotton genes encoding putative NAC domain proteins are preferentially expressed in roots and in responses to abiotic stress during root development. Plant Growth Regul, 71: 101–112.
[35]  Ichimura, K., Shinozaki, K., Tena, G., Sheen. J., Henry, Y., Champion, A., Kreis, M., Zhang, S., Hirt, H., Wilson, C. 2002. Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci, 7(7):301–308.
[36]  Jiang, W., Fu F.L., Zhang S.Z., Wu, L. and Li W.C. Cloning and characterization of functional trehalose-6-phosphate synthase gene in maize. J Plant Biol; 53(2): 134–141.
[37]  Jin L.G., Li H. and Liu J.Y., 2010. Molecular characterization of three ethylene responsive element binding factor genes from cotton, J integr Plant Biol, 52(5): 485-495.
[38]  Jin, L.G. and Liu, J.Y. 2008. Molecular cloning, expression profile and promoter analysis of a novel ethylene responsive transcription factor gene GhERF4 from cotton (Gossypium hirstum). Plant Physiol Biochem, 46:46–53.
[39]  Jonak, C., Kiegerl, S., Ligterink, W., Barker, P.J., Huskisson, N.S. and Hirt, H. 1996. Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought. Proc Natl Acad Sci USA, 93:11274–11279.
[40]  Kim, K.N., Cheong, Y.H., Gupta, R. and Luan, S. 2000. Interaction specificity of Arabidopsis calcineurin B-like calcium sensors and their target kinases. Plant Physiol, 124: 1844-1853.
[41]  Kistner, C. and Matamoros, M. 2005. Lotus japonicas Handbook: RNA isolation using phase extraction and LiCl precipitationm (Chapter 3.3). p.123-124.
[42]  Kohel, R.J. 1974. Influence of certain morphological characters on yield. Cotton Grow. Rev, 51: 281–292.
[43]  Koschützki, D. and Schreiber, F. 2008. Centrality analysis methods for biological networks and their application to gene regulatory networks. Gene Regul Syst Biol, 2:193–201.
[44]  Li, H.W., Zang, B.S., Deng, X.W. and Wang, X.P. 2011. Overexpression of the trehalose-6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice. Planta; 234 (5): 1007–1018.
[45]  Liu, J., Ishitani, M., Halfter, U., Kim, C.S. and Zhu, J.K. 2000. The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance. Proc Natl Acad Sci USA, 97: 3730–3734.
[46]  Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods, 25:402–408.
[47]  Martinez-Atienza, J., X., Jiang, B., Garciadeblas, I., Mendoza, J.K., Zhu, Pardo, J.M. and Quintero, F.J., 2007. Conservation of the salt overly sensitive pathway in rice. Plant Physiol, 143: 1001–1012.
[48]  Meng, C.M., Cai, C.P. and Zhang, T.Z., Guo, W.Z. 2009. Characterization of six novel NAC genes and their responses to abiotic stresses in Gossypium hirsutum L. Plant Sci, 176: 352–359.
[49]  Mikolajczyk, M., Awotunde, O.S., Muszynska, G., Klessig, D.F. and Dobrowolska, G., 2000. Osmotic stress induces rapid activation of a salicylic acid-induced protein kinase and a homolog of protein kinase ASK1 in tobacco cells. Plant Cell, 12:165–178.
[50]  Mu, M., Lu, X., Wang, J.J., Wang, D.L., Yin, Z.J., Wang, S., Fanand, W.L., Ye, W.W. 2016. Genome-wide Identification and analysis of the stress-resistance function of the TPS (Trehalose-6-PhosphateSynthase) gene family in cotton. BMC Genetics 17:54
[51]  Munnik, T., Ligterink, W., Meskiene, II., Calderini, O., Beyerly, J., Musgrave, A. and Hirt, H. 1999. Distinct osmo-sensing protein kinase pathways are involved in signaling moderate and severe hyperosmotic stress. Plant J, 20:381–388.
[52]  Munns R. 2005. Genes and salt tolerance: bringing them together, New Phytol, 167645–167663.
[53]  Munns, R. and Termaat, A. 1986. Whole-plant responses to salinity. Aus J Plant Physiol, 13, 143-160.
[54]  Osakabe Y, Mizuno S, Tanaka H, Maruyama K, Osakabe K, Todaka D, Fujita Y, Kobayashi M, Shinozaki K and Yamaguchi-Shinozaki K. 2010. Overproduction of the membrane-bound receptor-like protein kinase1, RPK1, enhances abiotic stress tolerance in Arabidopsis. J Biol Chem, 285: 9190–9201.
[55]  Ouyang, S.Q., Liu, Y.F., Liu, P., Lei, G., He, S.J., Ma, B., Zhang, W.K., Zhang, J.S.and Chen, S.Y. 2010. Receptor-like kinase OsSIK1 improves drought and salt stress tolerance in rice (Oryza sativa) plants. Plant J, 62:316–329.
[56]  Palmgren, M. G. 2001. Plant plasma membrane H+-ATPases: powerhouses for nutrient uptake. Annu Rev Plant Physiol Plant Mol Biol; 52: 817–45.
[57]  Peng Z., He S., Gong W., Sun J., Pan Z., Xu F., Lu Y., Du X. 2014. Comprehensive analysis of differentially expressed genes and transcriptional regulation induced by salt stress in two contrasting cotton genotypes. BMC Genomics, 15:760-772.
[58]  Pinney J.W. andWesthead D.R. 2006. Betweenness-based decomposition methods for social and biological networks. Interdiscip Stat Bioinf 87–90.
[59]  Shannon P, A. Markiel, O. Ozier, N. S. Baliga, J.T. Wang, D. Ramage, N. Amin, Schwikowski, B. and T. Ideker. 2003. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res, 13: 2498–2504
[60]  Shi, W., Hao, L., Li, J., Liu, D., Guo, X. and Li, H. 2014. The Gossypium hirsutum WRKY gene GhWRKY39-1 promotes pathogen infection defense responses and mediates salt stress tolerance in transgenic Nicotiana benthamiana. Plant Cell Rep, 33: 483–498.
[61]  Tanaka, H., Osakabe, Y., Katsura, S., Mizuno, S., Maruyama, K., Kusakabe, K., Mizoi, J., Shinozaki, K. and Yamaguchi-Shinozaki K. 2012. Abiotic stress-inducible receptor-like kinases negatively control ABA signal in Arabidopsis. Plant J, 70:599–613.
[62]  Tripathi, V., Parasuraman, B., Laxmi, A. and Chattopadhyay, D. 2009. CIPK6, a CBL-interacting protein kinase is required for development and salt tolerance in plants. Plant J. 58778–790.
[63]  Wijngaard, P.W., Sinnige, M.P., Roobeek, I., Reumer, A., Schoonheim, P.J., Mol, J.N., Wang, M. and and De Boer A.H. 2005. Abscisic acid and 14-3-3 proteins control K+ channel activity in barley embryonic root. Plant J; 41(1): 43–55.
[64]  Wu, C.A., Yang, G.D., Meng, Q.W. and Zheng, C.C., 2004. The Cotton GhNHX1 Gene Encoding a Novel Putative Tonoplast Na+/H+Antiporter Plays an Important Role in Salt Stress. Plant Cell Physiol. 45(5): 600–607.
[65]  Xiang, Y., Huang Y. and Xiong, L. 2007. Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement. Plant Physiol, 144:1416–1428.
[66]  Xiong, L. and Yang, Y. 2003. Disease resistance and abiotic stress tolerance in rice are inversely modulated by an abscisic acid-inducible mitogen-activated protein kinase. Plant Cell 15: 745–759.
[67]  Xue, T., Li, X., Zhu, W., Wu, C., Yang, G. and Zheng, C. 2009. Cotton metallothione in GhMT3a, a reactive oxygen species scavenger, increased tolerance against abiotic stress in transgenic tobacco and yeast. J Exp Bot, 60 339–349.
[68]  Yan, H., Jia, H., Chen, X., Hao, L., An, H. and Guo, X. 2014. The cotton WRKY transcription factor GhWRKY17 functions in drought and salt stress in transgenic Nicotiana benthamiana through ABA signaling and the modulation of reactive oxygen species production. Plant Cell Physiol. 55: 2060–2076.
[69]  Yan, J., He, C., Wang, J., Mao, Z., Holaday S.A., Allen, R.D. and Zhang H.2004. Overexpression of the Arabidopsis 14-3-3 protein GF14 lambda in cotton leads to a‘‘stay-green” phenotype and improves stress tolerance under moderate drought conditions. Plant Cell Physiol; 45(8): 1007–1014.
[70]  Yoon, J., Blumer, A. and Lee, K. 2006. An algorithm for modularity analysis of directed and weighted biological networks based on edge-betweenness centrality. Bioinformatics, 22:3106–3108.
[71]  Zentella, R, Mascorro-Gallardo, J.O., VanDijck, P., Folch-Mallol, J., Bonini, B., VanVaeck, C., Gaxiola, R., Covarrubias. A.A., Nieto-Sotelo, J., Thevelein, J.M. and Iturriaga, G. 1999. A Selaginella lepidophyll a trehalose-6-phosphate synthase complements growth and stress-tolerance defects in a yeast tps1 mutant. Plant Physiol.; 119(4): 1473–1482.
[72]  Zhang, H., Huang, Z., Xie, B., Chen, Q., Tian, X., Zhang, X., Zhang, H., Lu, X., Huang, D. and Huang, R. 2004. The ethylene, jasmonate, abscisic acid and NaCl-responsive tomato transcription factor JERF1 modulates expression of GCC box-containing genes and salt tolerance in tobacco. Planta 220, 262–270.
[73]  Zhang, L., Li, Y., Lu, W., Meng, F., Wu, C.A. and Guo, X. 2012. Cotton GhMKK5 affects disease resistance, induces HR-like cell death, and reduces the tolerance to salt and drought stress in transgenic Nicotiana benthamiana. J Exp Bot, 63: 3935–3951.
[74]  Zhang, L., Xi, D., Li, S., Gao, Z., Zhao, S., Shi, J., Wu, C. and Guo, X. 2011. A cotton group CMAP kinase gene, GhMPK2, positively regulates salt and drought tolerance in tobacco. Plant Mol Biol, 77: 17–31
[75]  Zhang, S. and Klessig, D.F. 1998. Resistance gene N-mediated de novo synthesis and activation of a tobacco mitogen-activated protein kinase by tobacco mosaic virus infection. Proc Natl Acad Sci USA, 95:7433–7438
[76]  Zhao, J., Gao, Y., Zhang, Z., Chen, T., Guo, W. and Zhang, T. 2013. A receptor-like kinase gene (GbRLK) from Gossypium barbadense enhances salinity and drought-stress tolerance in Arabidopsis, BMC Plant Biol. 13: 110–165.
[77] Zhao, J., Zhiyuan, Z., Yulong, G., Lei, Z., Lei, F., Xiangdong, C., Zhiyuan, N., Tianzi, C., Guo, W. and Zhang, T. 2015. Overexpression of GbRLK, a putative receptor-like kinase gene, improved cotton tolerance to Verticillium wilt. Sci Rep 5: 15048.