Abstract
Saline-alkali stress has adverse effects on plant growth. Some plant Na+/H+ antiporters were reported to be important in salt tolerance. However, it needs to be better understood that Na+/H+ antiporters are involved in plant salt-alkali (NaHCO3/Na2CO3) tolerance. In this study, a Na+/H+ antiporter gene LcNHX1 (China patent No.200810050629.1) has been cloned from Leymus chinensis. The LcNHX1 CDS contains 1614 bp that encodes 537 amino acids. Amino acid and nucleotide sequence similarity, protein topology modelling, conserved functional domains in the protein sequence, and subcellular localization classified LcNHX1 as a vacuolar NHX1 homolog. Transcription analysis by quantitative RT-PCR indicated that upregulated expression of LcNHX1 could be induced by NaCl, NaHCO3, NaCl + NaHCO3, and PGE in L. chinensis seedlings. The expression of LcNHX1 partially complements the salt-sensitive phenotypes of a Δnhx1 yeast strain. In addition, LcNHX1 overexpressing enhanced the tolerance to NaHCO3 stress in the transgenic Arabidopsis. Taken together, these results indicated that LcNHX1 is a potential candidate gene for enhancing plant saline-alkali tolerance.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Availability of Data and Materials
All data generated or analyzed during this study are included in this published article (and its supplementary information files).
References
Al-Harrasi I, Jana GA, Patankar HV, Al-Yahyai R, Rajappa S, Kumar PP, Yaish MW (2020) A novel tonoplast Na(+)/H(+) antiporter gene from date palm (PdNHX6) confers enhanced salt tolerance response in Arabidopsis. Plant Cell Rep 39:1079–1093. https://doi.org/10.1007/s00299-020-02549-5
Bart R, Chern M, Park CJ, Bartley L, Ronald PC (2006) A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2:13. https://doi.org/10.1186/1746-4811-2-13
Bassil E, Blumwald E (2014) The ins and outs of intracellular ion homeostasis: NHX-type cation/H(+) transporters. Curr Opin Plant Biol 22:1–6. https://doi.org/10.1016/j.pbi.2014.08.002
Bassil E, Tajima H, Liang YC, Ohto MA, Ushijima K, Nakano R, Esumi T, Coku A, Belmonte M, Blumwald E (2011) The Arabidopsis Na+/H+ antiporters NHX1 and NHX2 control vacuolar pH and K+ homeostasis to regulate growth, flower development, and reproduction. Plant Cell 23:3482–3497. https://doi.org/10.1105/tpc.111.089581
Bassil E, Zhang S, Gong H, Tajima H, Blumwald E (2019) Cation specificity of vacuolar NHX-type cation/H(+) antiporters. Plant Physiol 179:616–629. https://doi.org/10.1104/pp.18.01103
Brini F, Gaxiola RA, Berkowitz GA, Masmoudi K (2005) Cloning and characterization of a wheat vacuolar cation/proton antiporter and pyrophosphatase proton pump. Plant Physiol Biochem 43:347–354. https://doi.org/10.1016/j.plaphy.2005.02.010
Brini F, Hanin M, Mezghani I, Berkowitz GA, Masmoudi K (2007) 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. https://doi.org/10.1093/jxb/erl251
Cavusoglu E, Sari U, Tiryaki I (2023) Genome‐wide identification and expression analysis of Na+/H+antiporter (NHX) genes in tomato under salt stress. Plant Direct. https://doi.org/10.1002/pld3.543
Chauhan S, Forsthoefel N, Ran Y, Quigley F, Nelson DE, Bohnert HJ (2000) Na+/myo-inositol symporters and Na+/H+-antiport in Mesembryanthemum crystallinum. Plant J 24:511–522
Chen M, Chen QJ, Niu XG, Zhang R, Lin HQ, Xu CY, Wang XC, Wang GY, Chen J (2007) Expression of OsNHX1 gene in maize confers salt tolerance and promotes plant growth in the field. Plant. Soil and Environment - UZPI (czech Republic) 53:490–498
Ford BA, Ernest JR, Gendall AR (2012) Identification and characterization of orthologs of AtNHX5 and AtNHX6 in Brassica napus. Front Plant Sci 3:208. https://doi.org/10.3389/fpls.2012.00208
Galvez FJ, Baghour M, Hao G, Cagnac O, Rodriguez-Rosales MP, Venema K (2012) Expression of LeNHX isoforms in response to salt stress in salt sensitive and salt tolerant tomato species. Plant Physiol Biochem 51:109–115. https://doi.org/10.1016/j.plaphy.2011.10.012
Gao Y, Wang D, Ba L, Bai Y, Liu B (2008) Interactions between herbivory and resource availability on grazing tolerance of Leymus chinensis. Environ Exp Bot 63:113–122
Guan B, Hu Y, Zeng Y, Wang Y, Zhang F (2011) Molecular characterization and functional analysis of a vacuolar Na(+)/H(+) antiporter gene (HcNHX1) from Halostachys caspica. Mol Biol Rep 38:1889–1899. https://doi.org/10.1007/s11033-010-0307-8
Guan Q, Wang Z, Wang X, Takano T, Liu S (2015) A peroxisomal APX from Puccinellia tenuiflora improves the abiotic stress tolerance of transgenic Arabidopsis thaliana through decreasing of H2O2 accumulation. J Plant Physiol 175:183–191. https://doi.org/10.1016/j.jplph.2014.10.020
Han L, Xiao C, Xiao B, Wang M, Liu J, Bhanbhro N, Khan A, Wang H, Wang H, Yang C (2019) Proteomic profiling sheds light on alkali tolerance of common wheat (Triticum aestivum L.). Plant Physiol Biochem 138:58–64. https://doi.org/10.1016/j.plaphy.2019.02.024
He Y, Fu J, Yu C, Wang X, Jiang Q, Hong J, Lu K, Xue G, Yan C, James A et al (2015) Increasing cyclic electron flow is related to Na+ sequestration into vacuoles for salt tolerance in soybean. J Exp Bot 66:6877–6889. https://doi.org/10.1093/jxb/erv392
Hima Kumari P, Anil Kumar S, Ramesh K, Sudhakar Reddy P, Nagaraju M, Bhanu Prakash A, Shah T, Henderson A, Srivastava RK, Rajasheker G et al (2018) Genome-wide identification and analysis of Arabidopsis sodium proton antiporter (NHX) and human sodium proton exchanger (NHE) homologs in Sorghum bicolor. Genes (Basel). https://doi.org/10.3390/genes9050236
Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. Calif.agric.exp.stn.circ 347:357–359
Jha A, Joshi M, Yadav NS, Agarwal PK, Jha B (2011) Cloning and characterization of the Salicornia brachiata Na(+)/H(+) antiporter gene SbNHX1 and its expression by abiotic stress. Mol Biol Rep 38:1965–1973. https://doi.org/10.1007/s11033-010-0318-5
Jia B, Sun M, Sun X, Li R, Wang Z, Wu J, Wei Z, DuanMu H, Xiao J, Zhu Y (2016) Overexpression of GsGSTU13 and SCMRP in Medicago sativa confers increased salt-alkaline tolerance and methionine content. Physiol Plant 156:176–189. https://doi.org/10.1111/ppl.12350
Jiang X, Leidi EO, Pardo JM (2010) How do vacuolar NHX exchangers function in plant salt tolerance? Plant Signal Behav 5:792–795
Jin H, Plaha P, Park JY, Hong CP, Lee IS, Yang ZH, Jiang GB, Kwak SS, Liu SK, Lee JS (2006) Comparative EST profiles of leaf and root of Leymus chinensis, a xerophilous grass adapted to high pH sodic soil. Plant Sci 170:1081–1086
Jin H, Kim HR, Plaha P, Liu SK, Park JY, Piao YZ, Yang ZH, Jiang GB, Kwak SS, An G et al (2008) Expression profiling of the genes induced by Na2CO3 and NaCl stresses in leaves and roots of Leymus chinensis. Plant Sci 175:784–792. https://doi.org/10.1016/j.plantsci.2008.07.016
Jin S, Xu C, Li G, Sun D, Li Y, Wang X, Liu S (2017) Functional characterization of a type 2 metallothionein gene, SsMT2, from alkaline-tolerant Suaeda salsa. Sci Rep 7:17914. https://doi.org/10.1038/s41598-017-18263-4
Kagami T, Suzuki M (2005) Molecular and functional analysis of a vacuolar Na+/H+ antiporter gene of Rosa hybrida. Genes Genet Syst 80:121–128
Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845–858. https://doi.org/10.1038/nprot.2015.053
Kizhakkedath P, Jegadeeson V, Venkataraman G, Parida A (2015) A vacuolar antiporter is differentially regulated in leaves and roots of the halophytic wild rice Porteresia coarctata (Roxb.) Tateoka. Mol Biol Rep 42:1091–1105. https://doi.org/10.1007/s11033-014-3848-4
Li JY, He XW, Xu L, Zhou J, Wu P, Shou HX, Zhang FC (2008) Molecular and functional comparisons of the vacuolar Na+/H+ exchangers originated from glycophytic and halophytic species. J Zhejiang Univ Sci B 9:132–140. https://doi.org/10.1631/jzus.B0710445
Li R, Shi F, Fukuda K (2010) Interactive effects of various salt and alkali stresses on growth, organic solutes, and cation accumulation in a halophyte Spartina alterniflora (Poaceae). Environ Exp Bot 68:66–74
Li T, Tang S, Li W, Zhang S, Wang J, Pan D, Lin Z, Ma X, Chang Y, Liu B et al (2023) Genome evolution and initial breeding of the Triticeae grass Leymus chinensis dominating the Eurasian Steppe. Proc Natl Acad Sci USA 120:e2308984120. https://doi.org/10.1073/pnas.2308984120
Liu GS, Liu JS, Qi DC, Li HJ (2004) Factors affecting plant regeneration from tissue cultures of Chinese leymus (Leymus chinensis). Plant Cell, Tissue Organ Cult 76:175–178
Liu Q, Zhang Q, Burton RA, Shirley NJ, Atwell BJ (2010) Expression of vacuolar H+-pyrophosphatase (OVP3) is under control of an anoxia-inducible promoter in rice. Plant Mol Biol 72:47–60. https://doi.org/10.1007/s11103-009-9549-z
Liu L, Wang Y, Wang N, Dong YY, Fan XD, Liu XM, Yang J, Li HY (2011) Cloning of a vacuolar H(+)-pyrophosphatase gene from the halophyte Suaeda corniculata whose heterologous overexpression improves salt, saline-alkali and drought tolerance in Arabidopsis. J Integr Plant Biol 53:731–742. https://doi.org/10.1111/j.1744-7909.2011.01066.x
Liu Z, Meng H, Abdulla H, Zhang F, Mao X (2016) Cloning and characterization of metallothionein gene (HcMT) from Halostachys caspica and its expression in E. coli. Gene 585:221–227. https://doi.org/10.1016/j.gene.2016.03.039
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Mishra S, Alavilli H, Lee BH, Panda SK, Sahoo L (2014) Cloning and functional characterization of a vacuolar Na+/H+ antiporter gene from mungbean (VrNHX1) and its ectopic expression enhanced salt tolerance in Arabidopsis thaliana. PLoS ONE 9:e106678. https://doi.org/10.1371/journal.pone.0106678
Nass R, Cunningham KW, Rao R (1997) Intracellular sequestration of sodium by a novel Na+/H+ exchanger in yeast is enhanced by mutations in the plasma membrane H+-ATPase. Insights into mechanisms of sodium tolerance. J Biol Chem 272:26145–26152. https://doi.org/10.1074/jbc.272.42.26145
Peng YL, Gao ZW, Ying G, Liu GF, Sheng LX, Wang DL (2010) Eco-physiological characteristics of alfalfa seedlings in response to various mixed salt-alkaline stresses. J Integr Plant Biol 50:29–39
Pu L, Lin R, Zou T, Wang Z, Zhang M, Jian S (2021) Genome-wide identification, primary functional characterization of the NHX gene family in Canavalia rosea, and their possible roles for adaptation to tropical coral reefs. Genes (Basel) 1310.3390/genes13010033
Qiu QS (2012) Plant and yeast NHX antiporters: roles in membrane trafficking. J Integr Plant Biol 54:66–72. https://doi.org/10.1111/j.1744-7909.2012.01097.x
Qiu QS (2016) Plant endosomal NHX antiporters: activity and function. Plant Signal Behav 11:e1147643. https://doi.org/10.1080/15592324.2016.1147643
Quintero FJ, Blatt MR, Pardo JM (2000) Functional conservation between yeast and plant endosomal Na(+)/H(+) antiporters. FEBS Lett 471:224–228
Reguera M, Bassil E, Blumwald E (2014) Intracellular NHX-type cation/H+ antiporters in plants. Mol Plant 7:261–263. https://doi.org/10.1093/mp/sst091
Sato Y, Sakaguchi M (2005) Topogenic properties of transmembrane segments of Arabidopsis thaliana NHX1 reveal a common topology model of the Na+/H+ exchanger family. J Biochem 138:425–431. https://doi.org/10.1093/jb/mvi132
Sharma H, Taneja M, Upadhyay SK (2019) Identification, characterization and expression profiling of cation-proton antiporter superfamily in Triticum aestivum L. and functional analysis of TaNHX4-B. Genomics. https://doi.org/10.1016/j.ygeno.2019.02.015
Sharma P, Mishra S, Pandey B, Singh G (2023) Genome-wide identification and expression analysis of the NHX gene family under salt stress in wheat (Triticum aestivum L). Front Plant Sci. https://doi.org/10.3389/fpls.2023.1266699
Shen C, Yuan J, Li X, Chen R, Li D, Wang F, Liu X, Li X (2023) Genome-wide identification of NHX (Na+/H+ antiporter) gene family in Cucurbita L. and functional analysis of CmoNHX1 under salt stress. Front Plant Sci. https://doi.org/10.3389/fpls.2023.1136810
Shi D, Sheng Y (2005) Effect of various salt–alkaline mixed stress conditions on sunflower seedlings and analysis of their stress factors. Environ Exp Bot 54:8–21
Spyropoulos IC, Liakopoulos TD, Bagos PG, Hamodrakas SJ (2004) TMRPres2D: high quality visual representation of transmembrane protein models. Bioinformatics 20:3258–3260. https://doi.org/10.1093/bioinformatics/bth358
Wang J, Zuo K, Wu W, Song J, Sun X, Lin J, Li X, Tang K (2003) Molecular cloning and characterization of a new Na+/H+ antiporter gene from Brassica napus. DNA Seq 14:351–358
Wei Q, Hu P, Kuai BK (2012) Ectopic expression of an Ammopiptanthus mongolicus H + -pyrophosphatase gene enhances drought and salt tolerance in Arabidopsis. Plant Cell. Tissue Organ Cult 110:359–369
Whiteman SA, Serazetdinova L, Jones AM, Sanders D, Rathjen J, Peck SC, Maathuis FJ (2008) Identification of novel proteins and phosphorylation sites in a tonoplast enriched membrane fraction of Arabidopsis thaliana. Proteomics 8:3536–3547. https://doi.org/10.1002/pmic.200701104
Yang CW, Wang P, Li CY, Shi DC, Wang DL (2008) Comparison of effects of salt and alkali stresses on the growth and photosynthesis of wheat. Photosynthetica 46:107–114
Yang CW, Zhao N, Xu CM, Liu B, Shi DC (2012) Regulation of ion homeostasis in rice subjected to salt and alkali stresses. Aust J Crop Sci 6:724–731
Yao M, Zeng Y, Liu L, Huang Y, Zhao E, Zhang F (2012) Overexpression of the halophyte Kalidium foliatum H(+)-pyrophosphatase gene confers salt and drought tolerance in Arabidopsis thaliana. Mol Biol Rep 39:7989–7996. https://doi.org/10.1007/s11033-012-1645-5
Yarra R, Kirti PB (2019) Expressing class I wheat NHX (TaNHX2) gene in eggplant (Solanum melongena L.) improves plant performance under saline condition. Funct Integr Genomics https://doi.org/10.1007/s10142-019-00656-5
Ye CY, Zhang HC, Chen JH, Xia XL, Yin WL (2009) Molecular characterization of putative vacuolar NHX-type Na(+)/H(+) exchanger genes from the salt-resistant tree Populus euphratica. Physiol Plant 137:166–174. https://doi.org/10.1111/j.1399-3054.2009.01269.x
Yoshida K, Kawachi M, Mori M, Maeshima M, Kondo M, Nishimura M, Kondo T (2005) The involvement of tonoplast proton pumps and Na+(K+)/H+ exchangers in the change of petal color during flower opening of Morning Glory, Ipomoea tricolor cv. Heavenly Blue Plant Cell Physiol 46:407–415. https://doi.org/10.1093/pcp/pci057
Zeng Y, Li Q, Wang H, Zhang J, Du J, Feng H, Blumwald E, Yu L, Xu G (2018) Two NHX-type transporters from Helianthus tuberosus improve the tolerance of rice to salinity and nutrient deficiency stress. Plant Biotechnol J 16:310–321. https://doi.org/10.1111/pbi.12773
Zhang X, Henriques R, Lin SS, Niu QW, Chua NH (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1:641–646. https://doi.org/10.1038/nprot.2006.97
Zhang WD, Chen SY, Liu GS, Jan CC (2010) Seed-set and pollen-stigma compatibility in Leymus chinensis. Grass Forage Sci 59:180–185
Zhang YM, Zhang HM, Liu ZH, Li HC, Guo XL, Li GL (2015) The wheat NHX antiporter gene TaNHX2 confers salt tolerance in transgenic alfalfa by increasing the retention capacity of intracellular potassium. Plant Mol Biol 87:317–327. https://doi.org/10.1007/s11103-014-0278-6
Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6:66–71
Zhu D, Cai H, Luo X, Bai X, Deyholos MK, Chen Q, Chen C, Ji W, Zhu Y (2012) Over-expression of a novel JAZ family gene from Glycine soja, increases salt and alkali stress tolerance. Biochem Biophys Res Commun 426:273–279. https://doi.org/10.1016/j.bbrc.2012.08.086
Funding
This work was supported by the Jilin Scientific and Technological Development Program (20230101245JC).
Author information
Authors and Affiliations
Contributions
Y.Z. and J.G. conceived and designed the experiments. C.S. and Z.C. assembled sequences of NHX genes and performed phylogenetic analyses. C.S., Z.C., Y.W., and S.M. performed the transformation and subcellular localization assay. Y.L., Y.Y., Q.L., Z.W., and X.L. performed expression analysis of the LcNHX1. C.S., Z.C., Y.W., and S.M. carried out the salt tolerance assay, data collection, and analysis. Y.Z. and J.G. drafted the manuscript and generated the figures and tables. All authors reviewed the manuscript.
Corresponding authors
Ethics declarations
Ethical Approval
This manuscript is not applicable for both human and/or animal studies.
Competing Interests
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Sun, C., Zhang, C., Yin, Y. et al. Isolation and Functional Analysis of Na+/H+ Antiporter Gene (LcNHX1) from Leymus chinensis. Plant Mol Biol Rep (2024). https://doi.org/10.1007/s11105-024-01446-5
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11105-024-01446-5