Biologia plantarum 65:157-166, 2021 | DOI: 10.32615/bp.2021.003
Ectopic expression of GmNHX3 and GmNHX1, encoding two Glycine max Na+/H+ vacuolar antiporters, improves water deficit tolerance in Arabidopsis thaliana
- 1 Instituto de Tecnología Agroindustrial del Noroeste Argentino, Estación Experimental Agroindustrial Obispo Colombres - CONICET, Las Talitas, Tucumán, Argentina
- 2 Centro de Bioplantas, Universidad de Ciego de Ávila "Máximo Gómez Báez", Ciego de Ávila, Cuba
- 3 Facultad de Ciencias de la Universidad de la República, 11200 Montevideo, Uruguay
- 4 Instituto de Ciencia y Tecnología "Dr. César Milstein", CONICET, C1440FFX, Buenos Aires, Argentina
The importance of Na+/H+ antiporters in salt tolerance in plants has been demonstrated in many studies, but much less is known about their protective role during drought stress. To study their possible contribution to water deficit tolerance, two closely related soybean Na+/H+ antiporters belonging to the intracellular NHX exchanger protein family, GmNHX3 and GmNHX1, were evaluated in transgenic Arabidopsis thaliana. A. thaliana plants ectopically expressing GmNHX3 or GmNHX1 displayed a more drought-tolerant phenotype compared to wild-type plants, which was accompanied by an increase in relative water content and chlorophyll content during stress conditions. Both GmHNX1 and GmHNX3 transgenic lines accumulated higher amounts of Na+ and K+ cations, showed increased antioxidant enzyme activities and less membrane damage due to lipid peroxidation under water deficit, as compared to non-transformed plants. Furthermore, plants expressing GmNHX3 showed an increased sensitivity to abscisic acid as deduced from stomatal closure and seed germination inhibition studies. Finally, a significant up-regulation of abiotic stress-related genes was observed in both transgenic lines compared to wild-type plants in response to abscisic acid and mannitol treatments. These results demonstrate that GmNHX3 and GmNHX1 antiporters confer protection during drought stress in A. thaliana and hence are potential genetic targets to improve drought tolerance in soybean and other crops.
Keywords: abscisic acid, antioxidant enzymes, chlorophyll, drought tolerance, lipid peroxidation, mannitol, soybean, stomata.
Received: September 2, 2020; Revised: December 21, 2020; Accepted: January 12, 2021; Published online: June 9, 2021 Show citation
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References
- Adabnejad, H., Kavousi, H.R., Hamidi, H., Tavassolian, I.: Assessment of the vacuolar Na+/H+ antiporter (NHX1) transcriptional changes in Leptochloa fusca L. in response to salt and cadmium stresses. - Mol. Biol. Res. Commun. 4: 133-142, 2015.
- Antolín, M.C., Yoller, J., Sánchez-Díaz, M.: Effects of temporary drought on nitrate-fed and nitrogen-fixing alfalfa plants. - Plant Sci. 107: 159-165, 1995.
Go to original source... - Apse, M.P., Aharon, G.S., Snedden, W.A., Blumwald, E.: Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. - Science 285: 1256-1258, 1999.
Go to original source... - Baker, S.S., Wilhelm, K.S., Thomashow, M.F.: The 5'-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression. - Plant mol. Biol. 24: 701-713,1994.
Go to original source... - Bao, A.K., Du, B.Q., Touil, L., Kang, P., Wang, Q.-L., Wang, S.-M.: Co-expression of tonoplast cation/H+ antiporter and H+-pyrophosphatase from xerophyte Zygophyllum xanthoxylum improves alfalfa plant growth under salinity, drought and field conditions. - Plant Biotechnol. J. 14: 964-975, 2016.
Go to original source... - Bassil, E., Blumwald, E.: The ins and outs of intracellular ion homeostasis: NHX-type cation/H+ transporters. - Curr. Opin. Plant Biol. 22: 1-6, 2014.
Go to original source... - Bassil, E., Coku, A., Blumwald, E.: Cellular ion homeostasis: emerging roles of intracellular NHX Na+/H+ antiporters in plant growth and development. - J. exp. Bot. 63: 5727-5740, 2012.
Go to original source... - Bassil, E., Zhang, S., Gong, H., Tajima, H., Blumwald, E.: Cation specificity of vacuolar NHX-type cation/H+ antiporters. - Plant Physiol. 179: 616-629, 2019.
Go to original source... - Bates, L.S., Waldren, R.P., Teare, I.D.: Rapid determination of free proline for water-stress studies. - Plant Soil 39: 205-207, 1973.
Go to original source... - Beguerisse-Diaz, M., Hernandez-Gomez, M.C., Lizzul, A.M., Barahona, M., Desikan, R.: Compound stress response in stomatal closure: a mathematical model of ABA and ethylene interaction in guard cells. - BMC Systems Biol. 6:146, 2012.
Go to original source... - Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. - Anal. Biochem. 72: 248-254, 1976.
Go to original source... - Brini, F., Hanin, M., Mezghani, I., Berkowitz, G.A., Masmoudi, K.: Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants. - J. exp. Bot. 58: 301-308, 2007.
Go to original source... - Chance, B., Maehly, A.C.: Assay of catalases and peroxidases. Methods Enzymol. 2: 764-775, 1955.
Go to original source... - Clough, S.J., Bent, A.F.: Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. - Plant J. 16: 735-743,1998.
Go to original source... - Dong, H., Wang, C., Xing, C., Yang, T., Yan, J., Gao, J., Li, D., Wang, R., Blumwald, E., Zhang, S., Huang, X.: Overexpression of PbrNHX2 gene, a Na+/H+ antiporter gene isolated from Pyrus betulaefolia, confers enhanced tolerance to salt stress via modulating ROS levels.- Plant Sci. 285: 14-25, 2019.
Go to original source... - Fang, Y., Xiong, L.: General mechanisms of drought response and their application in drought resistance improvement in plants. - Cell. mol. Life Sci. 72: 673-689, 2015.
Go to original source... - Flowers, T.J., Munns, R., Colmer, T.D.: Sodium chloride toxicity and the cellular basis of salt tolerance in halophytes. - Ann. Bot. 115: 419-431, 2015.
Go to original source... - Fuganti-Pagliarini, R., Ferreira, L.C., Rodrigues, F.A., Molinari, H.B.C., Marin, S.R.R., Molinari, M.D.C., Marcolino-Gomes, J., Mertz-Henning, L.M., Farias, J.R.B., De Oliveira, M.C.N., Neumaier, N., Kanamori, N., Fujita, Y., Mizoi, J., Nakashima, K., Yamaguchi-Shinozaki, K., Nepomuceno, A.L.: Characterization of soybean genetically modified for drought tolerance in field conditions. - Front. Plant Sci. 8: 448, 2017.
Go to original source... - Galvez, F.J., Baghour, M., Hao, G., Cagnac, O., Rodriguez-Rosales, M.P., Venema, K.: Expression of LeNHX isoforms in response to salt stress in salt sensitive and salt tolerant tomato species. - Plant Physiol. Biochem. 51: 109-115, 2012.
Go to original source... - Garciarrubio, A., Legaria, J.P., Covarrubias, A.A.: Abscisic acid inhibits germination of mature Arabidopsis seeds by limiting the availability of energy and nutrients. - Planta 203: 182-187, 1997.
Go to original source... - Garde-Cerdán, T., López, R., Portu, J., González-Arenzana, L., López-Alfaro, I., Santamaría, P.: Study of the effects of proline, phenylalanine, and urea foliar application to Tempranillo vineyards on grape amino acid content. Comparison with commercial nitrogen fertilisers. - Food Chem. 163: 136-141, 2014.
Go to original source... - Gouiaa, S., Khoudi, H., Leidi, E.O., Pardo, J.M., Masmoudi, K.: Expression of wheat Na+/H+ antiporter TNHXS1 and H+- pyrophosphatase TVP1 genes in tobacco from a bicistronic transcriptional unit improves salt tolerance. - Plant mol. Biol. 79: 137-155, 2012.
Go to original source... - Grefen, C., Naomi, D., Kenji, H., Jörg, K., Schumacher, K., Blatt, M.R.: A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies. - Plant J. 64: 355-365, 2010.
Go to original source... - Gudesblat, G.E., Torres, P.S., Vojnov, A.A.: Xanthomonas campestris overcomes Arabidopsis stomatal innate immunity through a DSF cell-to-cell signal-regulated virulence factor. - Plant Physiol. 149: 1017-1027, 2009.
Go to original source... - Guo, Q., Tian, X.X., Mao, P.C., Meng, L.: Overexpression of Iris lactea tonoplast Na+/H+ antiporter gene IlNHX confers improved salt tolerance in tobacco. - Biol. Plant. 64: 50-57, 2020.
Go to original source... - Hodges, D.M., DeLong, J.M., Forney, C.F., Prange, R.F.: Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. - Planta 207: 604-611, 1999.
Go to original source... - Huertas, R., Rubio, L., Cagnac, O., Garcia-Sanchez, M.J., Alche, J.D., Venema, K., Fernandez, J.A., Rodriguez-Rosales, M.P.: The K+/H+ antiporter LeNHX2 increases salt tolerance by improving K+ homeostasis in transgenic tomato. - Plant Cell Environ. 36: 2135-2149, 2013.
Go to original source... - Jday, A., Rejeb, K.B., Slama, I., Saadallah, K., Bordenave, M., Planchais, S., Savouré, A., Abdelly, C.: Effects of exogenous nitric oxide on growth, proline accumulation and antioxidant capacity in Cakile maritima seedlings subjected to water deficit stress. - Funct. Plant Biol. 43: 939-948, 2016.
Go to original source... - Joshi, M., Jha, A., Mishra, A., Jha, B.: Developing transgenic Jatropha using the SbNHX1 gene from an extreme halophyte for cultivation in saline wasteland. - PloS ONE 8:e71136, 2013.
Go to original source... - Kar, M., Mishra, D.: Catalase, peroxidase, and polyphenoloxidase activities during rice leaf senescence. - Plant Physiol. 57: 315-319, 1976.
Go to original source... - Li, M., Li, Y., Li, H., Wu, G.: Overexpression of AtNHX5 improves tolerance to both salt and drought stress in Broussonetia papyrifera (L.) Vent. - Tree Physiol. 31: 349-357, 2011.
Go to original source... - Li, N., Wang, X., Ma, B., Du, C., Zheng, L., Wang, Y.: Expression of a Na+/H+ antiporter RtNHX1 from a recretohalophyte Reaumuria trigyna improved salt tolerance of transgenic Arabidopsis thaliana - J. Plant Physiol. 218: 109-120, 2017.
Go to original source... - Li, T., Zhang, Y., Liu, H., Wu, Y.T., Li, W., Zhang, H.: Stable expression of Arabidopsis vacuolar Na+/H+ antiporter gene AtNHX1, and salt tolerance in transgenic soybean for over six generations. - Chin. Sci. Bull. 55: 1127-1134, 2010.
Go to original source... - Li, W.Y., Wong, F.L., Tsai, S.N., Phang, T.H., Shao, G., Lam, H.M.: Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells. - Plant Cell Environ. 29: 1122-1137, 2006.
Go to original source... - Li, X.: Improved pyrogallol autoxidation method: a reliable and cheap superoxide-scavenging assay suitable for all antioxidants. - J. Agr. Food Chem. 60: 6418-6424, 2012.
Go to original source... - Ma, Y.C., Auge, R.M., Dong, C., Cheng, Z.M. : Increased salt tolerance with overexpression of cation/proton antiporter genes: a meta-analysis. - Plant biotechol. J. 15: 162-173, 2017.
Go to original source... - Maathuis, F.J., Ahmad, I., Patishtan, J.: Regulation of Na+ fluxes in plants - Front. Plant Sci. 5: 467, 2014.
Go to original source... - Mafakheri, A., Siosemardeh, A., Bahramnejad, B., Struik, P., Sohrabi, Y.: Effect of drought stress on yield, proline and chlorophyll contents in three chickpea cultivars. - Aust. J. Crop Sci. 4: 580-585, 2010.
- Mantyla, E., Lang, V., Palva, E.T.: Role of abscisic acid in drought-induced freezing tolerance, cold acclimation, and accumulation of LT178 and RAB18 proteins in Arabidopsis thaliana. - Plant Physiol. 107: 141-148, 1995.
Go to original source... - Marok, M.A., Tarrago, L., Ksas, B., Henri, P., Abrous-Belbachir, O., Havaux, M., Rey, P.: A drought-sensitive barley variety displays oxidative stress and strongly increased contents in low-molecular eight antioxidant compounds during water deficit compared to a tolerant variety. - J. Plant Physiol. 170: 633-645, 2013.
Go to original source... - Msanne, J., Lin, J., Stone, J.M., Awada, T.: Characterization of abiotic stress-responsive Arabidopsis thaliana RD29A and RD29B genes and evaluation of transgenes. - Planta 234: 97-107, 2011.
Go to original source... - Nakano, Y., Asada, K.: Purification of ascorbate peroxidase in spinach chloroplasts; its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. - Plant cell. Physiol. 28: 131-140, 1987.
- Ni, Z., Kim, E.D., Chen, Z.J.: Chlorophyll and starch assays. - Protocol Exchange 10: 1038, 2009.
Go to original source... - Ning, K.G., Shao, H., Yu, D.: Physiological and transcriptional responses to salt stress in salt-tolerant and salt-sensitive soybean (Glycine max [L.] Merr.) seedlings. - Land Degrad. Develop. 29: 2707-2019, 2018.
Go to original source... - Padmavathi, T., Rao, D.: Differential accumulation of osmolytes in 4 cultivars of peanut (Arachis hypogaea L.) under drought stress. - J. Crop Sci. Biotechnol. 16: 151-159, 2013.
Go to original source... - Pardo, E. M. (2015). Aplicaciones Biotecnológicas aplicadas al mejoramiento de la soja para la obtención de variedades con resistencia incrementada a estrés hídrico. Doctoral Thesis. Universidad Nacional de Tucumán. Tucumán, Argentina.
- Pardo, E.M., Vellicce, G., Aguirrezabal, L., Pereyra-Irujo, G., Rocha, C., García, M.G., Prieto Angueira, S., Welin, B., Sanchez, J., Ledesma, F.: Drought tolerance screening under controlled conditions predicts ranking of water-limited yield of field-grown soybean genotypes. - J. Agron. Crop Sci. 201: 95-104, 2015.
Go to original source... - Peleg, Z., Blumwald, E.: Hormone balance and abiotic stress tolerance in crop plants. - Curr. Opin. Plant Biol. 14: 290-295, 2011.
Go to original source... - Pfaffl, M.W.: A new mathematical model for relative quantification in real-time RT-PCR. - Nucl. Acids Res. 29: e45, 2001.
Go to original source... - Pyngrope, S., Bhoomika, K., Dubey, R.: Reactive oxygen species, ascorbate-glutathione pool, and enzymes of their metabolism in drought-sensitive and tolerant indica rice (Oryza sativa L.) seedlings subjected to progressing levels of water deficit. - Protoplasma 250: 585-600, 2013.
Go to original source... - Rahdari, P., Hoseini, S.M.: Drought stress: a review. - Int. J. Agron. Plant Prod. 3: 443-446, 2012.
- Ramakers, C., Ruijter, J.M., Deprez, R.H., Moorman, A.F.: Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. - Neurosci. Lett. 339: 62-66, 2003.
Go to original source... - Reguera, M., Bassil, E., Blumwald E: Intracellular NHX-type cation/H+ antiporters in plants. - Mol. Plants 7: 261-263, 2014.
Go to original source... - Di Rienzo, J.D.: fgStatistics, version 2009. Cordoba, Argentina, 2009.
- Di Rienzo, J. A.; Casanoves, F.; Balzarini, M. G.; Gonzalez, L.; Tablada,M.; Robledo, C. W. Infostat versin 2018. Córdoba, Argentina, 2018.
- Rodriguez-Gacio, M., Matilla-Vazquez, M.A., Matilla, A.J.: Seed dormancy and ABA signaling: the breakthrough goes on. - Plant Signal. Behav. 4: 1035-1049, 2009.
Go to original source... - Rodriguez-Rosales, M.P., Jiang, X., Galvez, F.J., Aranda, M.N., Cubero, B., Venema, K.: Overexpression of the tomato K+/H+ antiporter LeNHX2 confers salt tolerance by improving potassium compartmentalization. - New Phytol. 179: 366-377, 2008.
Go to original source... - Sekmen, A., Ozgur, R., Uzilday, B., Turkan, I.: Reactive oxygen species scavenging capacities of cotton (Gossypium hirsutum) cultivars under combined drought and heat induced oxidative stress. - Environ. exp. Bot. 99: 141-149, 2014.
Go to original source... - Sharp, R.E., Poroyko, V., Hejlek, L.G., Spollen, W.G., Springer, G.K., Bohnert, H.J., Nguyen, H.T.: Root growth maintenance during water deficits: physiology to functional genomics. - J. exp. Bot. 55: 2343-2351, 2004.
Go to original source... - Singh, A., Lawrence, K., Pandit, S., Lawrence, R.: Oxidative stress in leaves, stems and roots of Withania somnifera on copper exposure. - Int. J. Pharm. Sci. 30: 239-246, 2015.
- Strizhov, N., Abraham, E., Okresz, L., Blickling, S., Zilberstein, A., Schell, J., Koncz, C., Szabados, L.: Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis. - Plant J. 12: 557-569, 1997.
Go to original source... - Sun, Y., Wang, D.B., Yanling, W., Ningning, W.Y.: Studies on the overexpression of the soybean GmNHX1 in Lotus corniculatus: the reduced Na+ level is the basis of the increased salt tolerance. - Chin. Sci. Bull. 51: 1306-1315, 2006.
Go to original source... - Wang, H., Ding, Q., Wang, H.: A new Na+/H+ antiporter gene KvNHX1 isolated from the halophyte Kosteletzkya virginica improves salt tolerance in transgenic tobacco. - Biotech. Biotech. Equip. 32: 1378-1386, 2018.
Go to original source... - Wu, G.Q., Xi, J.J., Wang, Q., Bao, A.K., Ma, Q., Zhang, J.L, Wang, S.M.: The ZxNHX gene encoding tonoplast Na+/H+ antiporter from the xerophyte Zygophyllum xanthoxylum plays important roles in response to salt and drought. - J. Plant Physiol. 168: 758-767, 2011.
Go to original source... - Wu, H., Zhang, X., Giraldo, J., Shabala, S.: It is not all about sodium: revealing tissue specificity and signalling roles of potassium in plant responses to salt stress. - Plant Soil 431:1-17, 2018.
Go to original source... - Xiong, L., Ishitani, M., Zhu, J.K.: Interaction of osmotic stress, temperature, and abscisic acid in the regulation of gene expression in Arabidopsis. - Plant Physiol. 119: 205-212, 1999.
Go to original source... - Yamaguchi-Shinozaki, K., Shinozaki, K.: Arabidopsis DNA encoding two desiccation-responsive rd29 genes. - Plant Physiol. 101: 1119-1120, 1993a.
Go to original source... - Yamaguchi-Shinozaki, K., Shinozaki, K.: Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. - Mol. Gen. Genet. 236: 331-340, 1993b.
Go to original source... - Zhou, G., Guan, R., Li, Y., Chang, R., Qiu, L.: Molecular characterization of GmNHX2, a Na+/H+ antiporter gene homolog from soybean, and its heterologous expression to improve salt tolerance in Arabidopsis. - Chin. Sci. Bull. 54: 3536-3545, 2009.
Go to original source... - Zipper, S.C., Qiu, J., Kucharik, C.J.: Drought effects on US maize and soybean production: spatiotemporal patterns and historical changes. - Environ. Res. Lett. 11: 1-11, 2016.
Go to original source...



