Genome-Wide Identification, Structural Characterization and Phylogenetic Analysis of NHX (Na+/H+ Exchanger) in Lima Bean (Phaseolus Lunatus L.)

Arif Ullah; Zamarud Shah; Habiba Ali; Osaid Kamran Marwat; Kaleem Ullah

Genome-Wide Identification, Structural Characterization and Phylogenetic Analysis of NHX (Na+/H+ Exchanger) in Lima Bean (Phaseolus Lunatus L.)

Authors

Arif Ullah PhD Student, Department of Biotechnology, University of Science and Technology Bannu, Pakistan
Zamarud Shah Assistant Professor, Department of Biotechnology, Abdul Wali Khan University, Mardan, Pakistan
Habiba Ali BS Student, Department of Biotechnology, Abdul Wali Khan University, Mardan, Pakistan
Osaid Kamran Marwat M. Phil Student, School of Interdisciplinary Engineering and Sciences, National University of Science and Technology, Islamabad, Pakistan
Kaleem Ullah BS Student, Department of Biotechnology, Abdul Wali Khan University, Mardan, Pakistan

Keywords:

Lima bean, NHX, genome wide analysis, salt, tolerance

Abstract

The Na+/H+ exchanger (NHX), a gene family of membrane transporters, is well recognized for enhancing salt tolerance in plants. The NHXs assist in maintaining ion and pH homeostasis in cells by exchanging K+ or Na+ for H+. Genome-wide analysis of NHX genes has not been reported in lima bean. The current study aimed at exploring and characterizing the NHX gene family across the genome of the lima bean. Eight PlNHX genes in the genome of lima bean were detected during the present study, and named as PlNHX1-PlNHX8 on the basis of predicted sub-cellular localization. PlNHX8 was recorded as the largest gene while PlNHX5 was found as the smallest gene based on their CDS, protein length (PL) and protein molecular weight (PMW). Similarly, Isoelectric points and GRAVY were found in the range of 5.33 to 9.18 and 0.111 to 0.647, respectively. Vacoule was predicted as a major residence for PlNHX proteins. The varying number of exons reveals that events like deletion/addition have resulted in variation in motif number during the course of evolution, with motif3 conserved in all PlNHX genes. Phylogenetic tree reveals that paralogs contributed 25% to the PlNHX gene family expansion. Ka/Ks ratios of paralogs reveal that they were all under purifying selection. The promoter region has Box-4, G-box, ABRE and MeJA might be contributing elements for growth, development and defence functions of PlNHX. This study provides a basis for the functional validity of PlNHX genes.

References

Adebo, J. A. (2023). A review on the potential food application of lima beans (Phaseolus lunatus L.), an underutilized crop. Applied. Sciences. 13(3), 1996.

Aharon, G. S., Apse, M. P., Duan, S., Hua, X., & Blumwald, E. (2003). Characterization of a family of vacuolar Na +/H + antiporters in Arabidopsis thaliana. Plant and Soil, 253, 245–256

Ain-Ali, Q.U., Mushtaq, N., Amir, R., Gul, A., Tahir, M., Munir, F. (2021). Genome-wide promoter analysis, homology modelling and protein interaction network of dehydration-responsive element binding (DREB) gene family in Solanum tuberosum. PLoS One 16 (12), e0261215.

Akram, U., Song, Y., Liang, C., Abid, M.A., Askari, M., Myat, A.A., Abbas, M., Malik, W., Ali, Z., Guo, S., Zhang, R., Meng, Z. (2020). Genome-wide characterization and expression analysis of the NHX gene family under salinity stress in Gossypium barbadense and its comparison with Gossypium hirsutum. Genes (Basel) 11 (7), 803.

Apse, M.P., Aharon, G.S., Snedden, W.A., Blumwald, E., (1999). Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiporter in Arabidopsis. Science 285 (5431), 1256–1258.

Ashraf, M., & McNeilly, T. (2004). Salinity tolerance in Brassica oilseeds. CRC. Critical Reviews in Plant Sciences, 23, 157–174.

Ayadi, M., Martins, V., Ben Ayed, R., Jbir, R., Feki, M., Mzid, R., Géros, H., Aifa, S., & Hanana, M. (2020). Genome-wide identification, molecular characterization, and gene expression analyses of grapevine NHX antiporters suggest their involvement in growth, ripening, seed dormancy, and stress response. Biochemical Genetics, 58, 102–128.

Bailey, T. L., Williams, N, Misleh, C, & Li, WW (2006). MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Research, 34 (Web Server issue), W369-373

Bandeh-hagh, A., Toorchi, M., Mohammadi, A., Chaparzadeh, N., Salekdeh, G. H., & Kazemnia, H. (2008). Growth and osmotic adjustment of canola genotypes in response to salinity. Journal of Food Agriculture and Environment, 6, 201–208.

Bassil, E., Coku, A., Blumwald, E. (2012). Cellular ion homeostasis: emerging roles of intracellular NHX Na+/H+ antiporters in plant growth and development. Journal of Experimental. Botany. 63, 5727–5740.

Bassil, E., Ohto, M., Esumi, T., Tajima, H., Zhu, Z., Cagnac, O., Belmonte, M., Peleg, Z., Yamaguchi, T., & Blumwald, E. (2011). The Arabidopsis intracellular Na +/H + antiporters NHX5 and NHX6 are endosome-associated and necessary for plant growth and development. Plant Cell, 23, 224–239.

Baudoin, J. P., Rocha, O., Degreef, J., Maquet, A., & Guarino, L. (2006). Phaseolus lunatus L. Prota. 1.

Brett, C. L., Donowitz, M., & Rao, R. (2005). Evolutionary origins of eukaryotic sodium/proton exchangers. American Journal of Physiology - Cell Physiology, 288, C223–C239.

Chandna, R., Azooz, M. M., & Ahmad, P. (2013). Recent advances in metabolomics to reveal plant response during salt stress. In P. Ahmad, M. M. Azooz, & M. N. V. Prasad (Eds.), Salt stress in plants: Signalling, Omics and Adaptations (pp. 1–14). New York, NY: Springer. https:// doi.org/10.1007/978-1-4614-6108-1_1

Chen. C., Chen. H., He. Y., Xia. R. (2018) TBtools, a toolkit for biologists integrating various biological data handling tools with a user-friendly interface. BioRxiv, 289660.

Chen, H. T., Chen, X., Wu, B. Y., Yuan, X. X., Zhang, H. M., Cui, X. Y., & Liu, X. Q. (2015). Whole-genome identification and expression analysis of K+ + efflux antiporter (KEA) and Na +/H + antiporter (NHX) families under abiotic stress in soybean. Journal of Integrative Agriculture, 14, 1171–1183.

Chen, H.T., Chen, X., Wu, B.Y., Yuan, X.X., Zhang, H.M., Cui, X.Y., Liu, X.Q., 2015. Whole-genome Identification and expression analysis of K+ efflux antiporter (KEA) and Na+/H+ antiporter (NHX) families under abiotic stress in soybean. Journal of Integrative Agriculture. 14, 1171–118

Chutipaijit, S., Cha-um, S., & Sompornpailin, K. (2011). High contents of proline and anthocyanin increase protective response to salinity in Oryza sativa L. spp. indica. Australian Journal of Crop Science, 5, 1191–1198.

Counillon, L., Franchi, A., & Pouyssegur, J. (1993). A point mutation of the Na +/H + exchanger gene (NHE1) and amplification of the mutated allele confer amiloride resistance upon chronic acidosis. Proceedings of the National Academy of Sciences of the United States of America, 90, 4508–4512.

Deinlein, U., Stephan, A. B., Horie, T., Luo, W., Xu, G., & Schroeder, J. I. (2014). Plant salt-tolerance mechanisms. Trends in Plant Science, 19, 371–379.

Dong, J., Liu, C., Wang, Y., Zhao, Y., Ge, D., Yuan, Z., (2021). Genome-wide identification of the NHX gene family in Punica granatum L. and their expression patterns under salt stress. Agronomy 11 (2), 264.

Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R. D., Bairoch, A. (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Research. 31(13), 3784-3788

Gaxiola, R. A., Rao, R., Sherman, A., Grisafi, P., Alper, S. L., & Fink, G. R. (1999). The Arabidopsis thaliana proton transporters, AtNHX1 and Avp1, can function in cation detoxification in yeast. Proceedings of the National Academy of Sciences, 96, 1480–1485.

Goodstein, D. M., Shu, S., Howson. R. Neupane, R., Hayes, R.D., Fazo. J, Mitros T, Dirks W, Hellsten U, Putnam N, Rokhsar DS (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Research 40(D1):D1178–D1186

Halfter, U., Ishitani, M., & Zhu, J.-K. (2000). The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3. Proceedings of the National Academy of Sciences, 97, 3735–3740.

Hamamoto, S., Horie, T., Hauser, F., Deinlein, U., Schroeder, J. I., & Uozumi, N. (2015). HKT transporters mediate salt stress resistance in plants: From structure and function to the field. Current Opinion in Biotechnology, 32, 113–120.

Harrewijn, P. (1979). Potassium and plant health. Netherlands Journal of Plant Pathology, 85, 82.

Huang, L., Li, Z., Sun, C., Yin, S., Wang, B., Duan, T., Liu, Y., Li, J., & Pu, G. (2022). Genome-wide identification, molecular characterization, and gene expression analyses of honeysuckle NHX antiporters suggest their involvement in salt stress adaptation. PeerJ, 10, e13214.

Jin Z, Chandrasekaran U, Liu A (2014) Genome-wide analysis of the Dof transcription factors in castor bean (Ricinus communis L.). Genes & Genomics, 36(4), 527-537.

Kumar S, Stecher G, & Tamura K (2016). MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution 33(7), 1870-1874

Kumari, H., Kumar, S., Ramesh, K., Palakolanu, S.R., Marka, N., Prakash, A., Shah, T., Henderson, A., Srivastava, R., Rajasheker, G., 2018. Genome-wide identification and analysis of Arabidopsis sodium proton antiporter (NHX) and human sodium proton exchanger (NHE) homologs in Sorghum bicolor. Genes 9, 236.

Lescot, M., Déhais, P., Thijs, G., Marchal, K., Moreau, Y., Van de Peer, Y., Rombauts, S. (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Research 30(1): 325-327

Ling, L., Qu, Y., Zhu, J., Wang, D., Guo, C., 2020. Genome-wide identification and expression analysis of the VQ gene family in Cicer arietinum and Medicago truncatula. PeerJ. 8, e8471.

Liu, H., Wang, K., Mei, Q., Wang, X., Yang, J., Ma, F., & Mao, K. (2023). Genome-wide analysis of the Actinidia chinensis NHX family and characterization of the roles of AcNHX3 and AcNHX7 in regulating salt tolerance in Arabidopsis. Environmental and Experimental Botany, 214, 105477.

Liu, J., Ishitani, M., Halfter, U., Kim, C.-S., & Zhu, J.-K. (2000). The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance. Proceedings of the National Academy of Sciences, 97, 3730–3734.

López-Alcocer JDJ, Lépiz-Ildefonso R, González-Eguiarte DR, Rodríguez-Macías R, López-Alcocer E (2016). Morphological variability of wild Phaseolus lunatus L. from the western region of México. Revolution of Fitotec. Mex 39(1), 49-58

Luo, X., Yang, S., Luo, Y., Qiu, H., Li, T., Li, J., Chen, X., Zheng, X., Chen, Y., Zhang, J., Zhang, Z., & Qin, C. (2021). Molecular characterization and expression analysis of the Na +/H + exchanger gene family in Capsicum annuum L. Frontiers in Genetics, 12, 1547.

Mahajan, S., Pandey, G. K., & Tuteja, N. (2008). Calcium- and salt-stress signalling in plants: Shedding light on the SOS pathway. Archives of Biochemistry and Biophysics, 471, 146–158.

Manishankar, P., Wang, N., Köster, P., Alatar, A. A., & Kudla, J. (2018). Calcium signalling during salt stress and in the regulation of ion homeostasis. Journal of Experimental Botany, 69, 4215–4226.

Marchler-Bauer, A., Derbyshire, M.K., Gonzales, N.R., Lu, S., Chitsaz, F., Geer, L.Y., Hurwitz, D.I. (2015) CDD: NCBI's conserved domain database. 43(D1), D222-D226.

Marschner, H. (1995). Mineral nutrition of higher plants (2nd ed., pp. 6–78). London: Academic Press.

Martínez‐Castillo J, Camacho‐Pérez L, Villanueva‐Viramontes S, Andueza‐Noh RH, Chacón‐Sánchez M I (2014) Genetic structure within the Mesoamerican gene pool of wild Phaseolus lunatus (Fabaceae) from Mexico as revealed by microsatellite markers: implications for conservation and the domestication of the species. American Journal of Botany 101(5), 851-864.

Munns, R. (1993). Physiological processes limiting plant growth in saline soils: Some dogmas and hypotheses. Plant, Cell & Environment, 16, 15–24.

Munns, R., & Tester, M. (2008). Mechanisms of salinity tolerance. Annual Review of Plant Biology, 59, 651–681. https://doi.org/10.1146/ annurev. A plant.59.032607.092911

Nascimento MDGR, Alves EU, Silva ML, Rodrigues C (2017) Lima bean (Phaseolus lunatus L.) seeds exposed to different salt concentrations and temperatures. Revolution of Caatinga, 30, 738-747.

Nataraja, K. N., & Parvathi, M. S. (2016). Tolerance to drought stress in plants: Unravelling the signalling networks. Drought Stress Tolerance in Plants, 5, 71–90.

Pandolfi, C., Mancuso, S., & Shabala, S. (2012). Physiology of acclimation to salinity stress in pea (Pisum sativum). Environmental and Experimental Botany, 84, 44–51.

Pardo, J. M., Cubero, B., Leidi, E. O., & Quintero, F. J. (2006). Alkali cation exchangers: Roles in cellular homeostasis and stress tolerance. Journal of Experimental Botany, 57, 1181–1199.

Parihar, P., Singh, S., Singh, R., Singh, V. P., & Prasad, S. M. (2015). Effect of salinity stress on plants and their tolerance strategies: A review. Environmental Science and Pollution Research, 22, 4056–4075.

Parveen, K., Saddique, M. A. B., Rehman, S. U., Ali, Z., Aziz, I., Shamsi, I. H., & Muneer, M. A. (2023). Identification and characterization of salt stress-responsive NHX gene family in chickpea. Plant Stress, 10, 100266.

Paul, A., Chatterjee, A., Subrahmanya, S., Shen, G., Mishra, N., 2021. NHX gene family in Camellia sinensis: in-silico genome-wide identification, expression profiles, and regulatory network analysis. Frontier of Plant Sciences. 12, 777884.

Pehlivan, N., Sun, L., Jarrett, P., Yang, X., Mishra, N., Chen, L., Kadioglu, A., Shen, G., Zhang, H., 2016. Co-overexpressing a plasma membrane and a vacuolar membrane sodium/proton antiporter significantly improves salt tolerance in transgenic Arabidopsis plants. Plant Cell Physiology. 57, 1069–1084.

Qiu, Q. S., Guo, Y., Dietrich, M. A., Schumaker, K. S., & Zhu, J. K. (2002). Regulation of SOS1, a plasma membrane Na +/H + exchanger in Arabidopsis thaliana, by SOS2 and SOS3. Proceedings of the National Academy of Sciences of the United States of America, 99, 8436–8441.

Qiu, Q.S., 2012. Plant and yeast NHX antiporters: roles in membrane trafficking. Journal of Integrative Plant Biology. 54, 66–72.

Sá FVS, Oliveira FS, Torres SB, Paiva EP, Nogueira NW, Sarmento ECS, Melo, AS (2021) Hydric and saline stress on Phaseolus lunatus L. seeds. Brazilian Journal of Biology. 82, e233550.

Sandhu, D., Pudussery, M. V., Kaundal, R., Suarez, D. L., Kaundal, A., & Sekhon, R. S. (2018). Molecular characterization and expression analysis of the Na +/H + exchanger gene family in Medicago truncatula. Functional & Integrative Genomics, 18, 141–153.

Shabala, S., & Cuin, T. A. (2008). Potassium transport and plant salt tolerance. Physiologia Plantarum, 133, 651–669.

Shah, A.A., Aslam, S., Akbar, M., Ahmad, A., Khan, W.U., Yasin, N.A., Ali, S, 2021. Combined effect of Bacillus fortis IAGS 223 and zinc oxide nanoparticles to alleviate cadmium phytotoxicity in Cucumis melo. Plant Physiology & Biochemistry. 158, 1–12.

Shi, H., Lee, B., Wu, S. J., & Zhu, J. K. (2003). Overexpression of a plasma membrane Na +/H + antiporter gene improves salt tolerance in Arabidopsis thaliana. Nature Biotechnology, 21, 81–85.

Solis, C.A., Yong, M.T., Zhou, M., Venkataraman, G., Shabala, L., Holford, P., Shabala, S., Chen, Z.H., 2022. Evolutionary significance of the NHX family and NHX1 in salinity stress adaptation in the genus Oryza. International Journal of Molecular Sciences 23, 2092.

Tester, M., & Davenport, R. (2003). Na+ tolerance and Na+ transport in higher plants. Annals of Botany, 91, 503–527.

Tian, F., Chang, E., Li, Y., Sun, P., Hu, J., & Zhang, J. (2017). Expression and integrated network analyses revealed functional divergence of NHXtype Na +/H + exchanger genes in poplar. Scientific Reports, 7, 1–17.

Valiollah, R. (2013). Effect of salinity stress on yield, component characters and nutrient compositions in rapeseed (Brassica napus L.) genotypes. Agricultura Tropica et Subtropica, 46, 58–63.

Van Zelm, E., Zhang, Y., & Testerink, C. (2020). Salt tolerance mechanisms of plants. Annual Review of Plant Biology, 71, 403–433.

Wang, L., Wu, X., Liu, Y., Qiu, Q.S., 2015. AtNHX5 and AtNHX6 control cellular K+ and pH homeostasis in Arabidopsis: three conserved acidic residues are essential for K+ transport. PLoS One 10 (12), e0144716.

Wang, Y., Mostafa, S., Zeng, W., Jin, B., 2021c. Function and mechanism of jasmonic acid in plant responses to abiotic and biotic stresses. International Journal of Molecular Sciences 22 (16), 8568.

Ward, J. M., Mäser, P., & Schroeder, J. I. (2009). Plant ion channels: Gene families, physiology, and functional genomics analyses. Annual Review of Physiology, 71, 59–82.

Wolfe D, Dudek S, Ritchie MD, & Pendergrass SAJBm (2013). Visualizing genomic information across chromosomes with PhenoGram. 6(1), 1-12.

Wu, G. Q., Wang, J. L., & Li, S. J. (2019). Genome-wide identification of Na +/H + antiporter (NHX) genes in sugar beet (Beta vulgaris L.) and their regulated expression under salt stress. Genes (Basel), 10, 401.

Yamaguchi, T., Apse, M. P., Shi, H., & Blumwald, E. (2003). Topological analysis of a plant vacuolar Na +/H + antiporter reveals a luminal C terminus that regulates antiporter cation selectivity. Proceedings of the National Academy of Sciences of the United States of America, 100, 12510–12515.

Yamaguchi, T., Hamamoto, S., & Uozumi, N. (2013). Sodium transport system in plant cells. Frontiers in Plant Science, 4, 140–151.

Zhang, H., Han, B., Wang, T., Chen, S., Li, H., Zhang, Y., Dai, S., (2012). Mechanisms of plant salt response: insights from proteomics. Journal of Proteome Research. 11 (1), 49–67.

Zhang, M., Qin, Z., & Liu, X. (2005). Remote-sensed spectral imagery to detect late blight in field tomatoes. Precision Agriculture, 6, 489–508.

Genome-Wide Identification, Structural Characterization and Phylogenetic Analysis of NHX (Na+/H+ Exchanger) in Lima Bean (Phaseolus Lunatus L.)