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The wheat NHX gene family: Potential role in improving salinity stress tolerance of plants
Plant Gene 18 (2019) 100178
Contents lists available at ScienceDirect
Plant Gene journal homepage: www.elsevier.com/locate/plantgene
The wheat NHX gene family: Potential role in improving salinity stress tolerance of plants
T
Rajesh Yarra State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
A R T I C LE I N FO
A B S T R A C T
Keywords: NHX antiporters Salinity stress Vegetable crops Forage legumes
Abiotic stress such as salinity adversely influences the growth, development and yield productivity of crop plants. Development of transgenic crops resilient to salinity stress is indispensable to ensure food security around the globe. Identification and functional validation of novel genes during abiotic stress adaptation of plants will afford the basis for effective genetic engineering approaches to enhance salinity stress tolerance of crop plants. The membrane and vacuolar Na+/H+ antiporters provide the best mechanism for ionic homeostasis in plants under salt stress. The function of vacuolar NHX antiporters in plants has been characterized and expressed in heterologous systems to enhance salinity stress tolerance. To date, a total of three vacuolar localized NHX genes have been identified from the wheat genome. Among the three wheat vacuolar NHX antiporters, TaNHX2 plays a vital role in conferring salinity tolerance in various crop plants. In this review, the author has discussed the potential role of wheat NHX genes in conferring salinity tolerance in Arabidopsis, tobacco, vegetable, and legume forage crops via genetic engineering approach. Finally, proposed the future prospects to engineer the higher plants genome with wheat NHX genes for sustainable food production in salinity affected areas.
1. Introduction The extent of agricultural farming area affected by high salinity is increasing day by day around the globe and about 20% of irrigated arable land is already under threaten (Qadir et al., 2014; Munns and Tester, 2008). Salinity stress is one of the most threatening environmental constraints that negatively affects growth, development and overall productivity of plants through induction of ion toxicity, water uptake deficiency, hormonal disturbance and oxidative stress (Ahanger et al., 2017; Ashraf and Foolad, 2013; Munns, 2005). To cope with the high salinity induced effects, plants trigger several stress tolerance responses including compartmentation, exclusion, and secretion of Na+/ H+ ions into vacuoles (Ahanger et al., 2017; Arzani, 2016). The unique structural feature of plant cells is the presence of vacuoles. Plant Na+/ H+ exchangers (NHXs) are intracellular membrane proteins that play a major role in pH, K+ and Na+ homeostasis of cells in higher plants. Most of the Na+/H+ antiporters are localized in the vacuole and involved in sequestration of Na+ within the vacuole under stressful conditions (Jia et al., 2018; Apse et al., 1999, 2003). These vacuolar antiporters regulate the exchange of H+/Na+ across tonoplast to reduce the toxicity of Na+ concentration in the cytoplasm (Arzani, 2016). The vacuolar Na+/H+ antiporters mediate the sequestration of excessive Na+ under salt stress (Blumwald and Poole, 1985, 1987) and
facilitate Na+ uptake into vacuoles, which is driven by the vacuolar proton gradient established by the vacuolar (V-type) proton ATPase that acidifies the vacuolar lumen (Fig. 1). The subsequent vacuolar Na+ sequestration protects important enzymatic reactions in the cytoplasm from excess Na+ levels while maintaining turgor (Horie and Schroeder, 2004). Among the identified NHX genes in higher plants, the wheat NHX genes (TaNHX1, TaNHX2 & TaNHX3) have a prospective role in improving the salt tolerance of various crop plants (Table 1). These three wheat NHX genes were firstly isolated by screening the wheat cDNA library (Brini et al., 2005; Lu et al., 2014; Yu et al., 2007). Under the saline condition, the expression of TaNHX2 and TaNHX3 transcripts is higher in leaves and roots of wheat plants (Lu et al., 2014; Yu et al., 2007) where TaNHX1 expression is higher only in roots compared to leaves (Brini et al., 2005). All these three wheat NHX proteins contain 12 membrane-spanning domains (Fig. 2) and one highly conserved amiloride binding domain (LFFIYLLPPI motif; amino acid residue 86–96) (Fig. 3). These transmembrane domains of wheat NHX proteins are highly conserved with regard to other NHX proteins of plants and responsible for targeting them to tonoplast. The transgenic technology is an important approach for developing salt-tolerant crops to withstand adverse environmental conditions (Dhankher and Foyer, 2018). The crucial step before proceeding with transgenic crops is the identification of potential candidate genes
E-mail address: rajeshyarra@rediffmail.com. https://doi.org/10.1016/j.plgene.2019.100178 Received 18 January 2019; Received in revised form 12 March 2019; Accepted 13 March 2019 Available online 14 March 2019 2352-4073/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Schematic representation showing Na+/H+ exchange across tonoplast and plasma membrane. Excess Na+ is secreted into the vacuole through NHXs (NHX1/NHX2/NHX3). The proton gradient across the vacuolar membrane is established by H+pyrophosphatase (AVP1) and the vacuolar H+ATPase (V- ATPase). The proton gradient across the plasma membrane is established through PMATPase.
yeast transformants expressing TaNHX1 complement yeast nhx1 sensitivity to cationic antibiotic hygromycin (Brini et al., 2005). The yeast transformants expressing TaNHX2, TaNHX3 showed better growth and survival rate compared to control under 400 mM NaCl and 200 mM NaCl stress conditions respectively. These results revealed that wheat NHX genes can complement the yeast mutant deficient in vacuolar Na+/H+ antiporter exchanging activities for better survival under salt stress conditions.
serving as key regulators of different metabolic pathways, including osmolyte synthesis, ion homeostasis through selective ion uptake (Ahanger et al., 2017). There are various NHX genes that might be utilized to transform crops to enhance salinity tolerance. In this review, I focus on the three important wheat NHX genes that greatly improve salinity tolerance in various plants. A complement analysis of wheat NHX genes in nhx yeast mutants confirmed their role under salt stress conditions (Brini et al., 2005; Lu et al., 2014; Yu et al., 2007). Further studies revealed that the ectopic expression of TaNHX1 and TaNHX3 in tobacco improves the growth performance under saline conditions (Brini et al., 2007; Lu et al., 2014) (Table 1). Till now, the effect of TaNHX1 and TaNHX3 expression is only reported in transgenic tobacco plants under salt stress conditions (Table 1). But functional validation of TaNHX2 expression is highly explored in transgenic vegetable plants and forage legume crops (Table 1). Especially, our research groups have been working in China and India since 2011 in this research area and successfully generated transgenic vegetable plants (Eggplant, Chill pepper and Tomato) tolerant to high salinity stress with TaNHX2 gene through genetic engineering approach. In this review, I summarize the role of these potential candidate genes (TaNHX1, TaNHX2 & TaNHX3) for improving the salt tolerance in various plants with special emphasis on major vegetable plants (Eggplant, Chilli pepper, and Tomato) and forage legume crops (soybean, alfalfa, and L. corniculatus).
3. Enhanced salt tolerance in transgenic plants expressing wheat NHX genes 3.1. TaNHX1 improves salt tolerance in A. thaliana plants Brini et al. (2005) reported that expression of TaNHX1 in Arabidopsis plants significantly improves the salinity tolerance of transgenic plants under salt stress conditions (200 mM NaCl). The Arabidopsis transgenic plants were generated by Agrobacterium-mediated transformation using binary vector pCB302.2 that contains TaNHX1 and TVP1 with a tandem repeat of the 35S promoter, the 35S terminator, and the BAR gene for resistance between the NOS promoter and terminator. The data have shown that the greater accumulation of Na+/K+ contents in the leaves of transgenic Arabidopsis plants compared to wild type plants under salt stress conditions. Moreover, TaNHX1 expressing Arabidopsis plants also displayed greater stability of relative water content compared with wild type plants under salt stress conditions. Taken together, all these results clearly indicate that TaNHX1 plays an important role in improving the salinity stress tolerance of transgenic plants under high NaCl conditions.
2. Complement analysis of wheat NHX genes in yeast transformants The expression of wheat NHX genes namely, TaNHX1, TaNHX2, and TaNHX3 in salt-sensitive yeast mutants (nhx1: W303) improved the survival rate of yeast mutants under salt stress conditions. The pRS8 plasmid harboring TaNHX1 under the control PMA1 promoter was used to generate yeast transformants (Brini et al., 2005). A yeast expression vectors pYES2 harboring TaNHX2 (pYES2-TaNHX2) (Yu et al., 2007) and TaNHX3 (pYES2-TaNHX3) (Lu et al., 2014) under the control of GAL1 promoter, were used to generate yeast transformants. These binary cassette plasmids and the control plasmids (pRS8, pYES2) were subjected to transform nhx1: W303, a yeast strain with a deficient function in the vacuolar Na+/H+ antiporter exchanging activity. The
3.2. TaNHX3 improves salt tolerance in tobacco plants Lu et al. (2014) generated the transgenic tobacco plants with enhanced salinity tolerance via overexpressing TaNHX3 gene. The binary plasmid pCAMBIA3301 containing TaNHX3 gene was used to generate transgenic tobacco plants through Agrobacterium-mediated transformation. The salt tolerance and stress associated physiological processes were significantly improved in transgenic tobacco plants. Transgenic plants accumulated more Na+ contents, higher fresh and dry weights, 2
Plant Gene 18 (2019) 100178
Zhang et al. (2012) Jian et al. (2009) Lu et al. (2014) Transgenic alfalfa plants were tolerant to 200 mM NaCl stress Transgenic plants were successfully grown in 150 mM NaCl stress Transgenic tobacco plants were tolerant to 150 mM NaCl stress Alfalfa (cv.Sandeli) L. corniculatus Tobacco (cv.Wisconsin38) TaNHX2 TaNHX2 TaNHX3
Brini et al. (2007) Yarra and Kirti (2019) Bulle et al. (2016) Yarra et al. (2012) Cao et al. (2011)
Floral buds Leaves Cotyledons Cotyledons Roots, Cotyledons Cotyledonary nodes Cotyledons Roots Leaves
Arabidopsis thaliana (eco:col) Eggplant (cv.PPL) Chilli Pepper (cv.G4) Tomato (cv. PED) Soyabean (cv.Zigongdongdou)
Transgenic plants grew well under 200 mM NaCl stress Transgenic eggplants exhibited an increased tolerance levels to 200 mM NaCl stress Transgenic Cilli Pepper plants were found to be tolerant against 200 mM NaCl stress Improved the salinity tolerance in transgenic tomato plants against 150 mM NaCl stress Higher level of salt tolerance was found in the transgenic lines against 200 mM and 150 mM NaCl stress
Vegetables play a vital role in human nutrition as they are rich sources for minerals, micronutrients, vitamins, antioxidants, phytosterols, and dietary fiber. Vegetable crop cultivation is also an imperative need to boost the agricultural economy for many countries (Dalal et al., 2006). The solanaceae or nightshade family of vegetables includes tomato, eggplant, and chilli pepper are indispensable for the human diet because of their nutritional value. Salinity stress causes considerable loss of these important vegetable crops productivity and quality in abiotic stress-prone areas (Zhuang et al., 2014; Bulle et al., 2016; Bai et al., 2018). Development of transgenic vegetable crops with an inherent capacity to withstand salinity stress would help in alleviating the vegetable production for sustainable food production and security. Selection of a suitable candidate gene is an important strategy to develop salt-tolerant transgenic plants. In hunt of a suitable candidate gene, we have chosen wheat TaNHX2 gene to develop the transgenic eggplant, chilli pepper, and tomato plants tolerant to salinity stress. Since 2011, we have been trying to achieve the goal and finally produced the salt tolerant eggplant, chilli pepper, and tomato plants through genetic engineering approach (Yarra and Kirti, 2019; Bulle et al., 2016; Yarra et al., 2012). These reports proved the potentiality of TaNHX2 gene for improving the salt tolerance of transgenic vegetable plants. We have used transformation vector pBIN438 harboring TaNHX2 gene, driven by the double 35S promoter and NPTII as a selection marker for the transformation of vegetable plants. Recently, we have published a report on the successful production of transgenic eggplants tolerant to salt stress (200 mM NaCl) by introducing TaNHX2 gene in eggplant (cv.PPL) genome via Agrobacteriummediated genetic transformation (Yarra and Kirti, 2019). Integration and expression of TaNHX2 transcripts in transgenic eggplants were confirmed by RT-PCR, qRT-PCR and southern hybridization. Transgenic eggplants (T2) expressing TaNHX2 displayed improved growth performance, stable leaf relative water content and chlorophyll content, proline accumulation, improved photosynthetic efficiency, transpiration rate, stomatal conductivity than the non-transformed plants under salinity stress (200 mM NaCl). The eggplant transgenics also showed decreased MDA content, hydrogen peroxide, oxygen radical production accompanying with the significant increase of antioxidant enzymes (SOD, APX, GPX, and GR) activity than non-transformed plants under salt stress (200 mM NaCl). The adaptation of plants to salinity stress is determined by biochemical alternations that enable the retention of water content, maintenance of ion homeostasis and photosynthetic apparatus protection (Dhankher and Foyer, 2018). The free radical scavenging capacity of the plants under salinity stress is an essential need. TaNHX2 expression in eggplants also triggered the biochemical pathways such as ion homeostasis, radical scavenging capacity and increased level of antioxidant enzymes for the survival under salinity conditions without any growth defects (Fig. 4). Bulle et al. (2016) published a report on transgenic chill pepper plants tolerant to salinity stress. We have successfully overcome the various barriers to achieve the genetic transformation of chilli pepper (cv.G4) plants via the introduction of TaNHX2 gene through Agrobacterium-mediated genetic transformation. Integration and expression of the TaNHX2 gene in chilli pepper plants genome is confirmed by RTPCR and southern hybridization. T1 transgenic plants exhibited better
tumefaciens tumefaciens (LBA4404) tumefaciens (LBA4404) tumefaciens (LBA4404) rhizogenes (K599) tumefaciens (EHA101) tumefaciens (EHA105) rhizogenes (K599) tumefaciens (EHA105) Agrobacterium Agrobacterium Agrobacterium Agrobacterium Agrobacterium Agrobacterium Agrobacterium Agrobacterium Agrobacterium
TaNHX1 TaNHX2 TaNHX2 TaNHX2 TaNHX2
References Character/trait improved Plant Explants
Gene
more accumulative nitrogen, phosphorus and potassium, higher contents of chlorophyll, carotenoid, soluble protein. Transgenic tobacco plants also displayed a significant increase of the antioxidant enzyme activities including superoxide dismutase, catalase, and peroxidase associated with the lower amounts of malondialdehyde and H2O2 compared to wild type plants. These results clearly demonstrated that TaNHX3 also play an important regulatory role in ROS scavenging capacity under salt stress conditions (Fig. 4). 3.3. TaNHX2 improves salt tolerance in transgenic vegetable plants (eggplant, chilli pepper, tomato)
Method of transformation
Table 1 Salt tolerant transgenic plants developed so far by introducing wheat NHX genes.
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Fig. 2. Diagram showing the twelve conserved transmembrane domains of wheat NHX proteins (TaNHX1, TaNHX2, and TaNHX3).
forage legume crops is confronting with adverse environmental conditions including salinity stress, indicating the need for tolerant varieties that can withstand and acclimatize to the deleterious effects of salinity stress. Advancements in genetic engineering facilitated to develop salinity tolerant varieties by introducing heterologous salt responsive candidate genes. In view of this, researchers introduced wheat NHX gene in some forage legume crops and developed salt-tolerant transgenic crops, which includes soybean, alfalfa, L. corniculatus (Cao et al., 2011; Zhang et al., 2012; Jian et al., 2009). Soybean (Glycine max (L.) Merrill) is an important plant source for the human diet as a protein resource and also used as an oil crop throughout the world. Salinity stress has adverse effects on seed germination, vegetative and reproductive growth of soybean plants (Shu et al., 2017). The improvement of soybean by genetic engineering is needed to withstand the salinity stress damage on soybean plants. Cao et al. (2011) reported the transformation of soybean plants (cv. Zigongdongdou) with TaNHX2 gene using the Agrobacterium-mediated transformation method. They have performed hairy root transformation as well as whole plant transformation via A. rhizogenes and A. tumefaciens using the binary vectors pGUS-TaNHX2, pTF101.1-TaNHX2 respectively. Interestingly, overexpression of TaNHX2 in soybean enhanced salt tolerance of transgenic hairy roots as well as composite and whole transgenic plants against 150 mM NaCl stress conditions. Alfalfa (Medicago sativa L.) is a crucial global perennial leguminous forage crop in temperate regions. Alfalfa crop productivity is adversely affected by salinity present in the soil (Rahman et al., 2015). Dissecting
growth performance under 200 mM NaCl conditions compared to wild type plants. Biochemical parameters assayed to analyze the salt tolerance capacity of transgenic plants under stressful conditions. Transgenic plants expressing TaNHX2 displayed increased levels of chlorophyll content, relative water content, superoxide dismutase, ascorbate peroxidase and reduced levels of hydrogen peroxide, malondialdehyde contents compared to wild type plants. Yarra et al. (2012) reported the successful transformation of tomato (cv.PED) plants with TaNHX2 gene through Agrobacterium-mediated approach. The transgenic tomato plants (T1) showed enhanced growth performance under 150 mM NaCl stress conditions compared to wild type plants. Integration and expression of TaNHX2 gene in the tomato genome is confirmed by RT-PCR and southern hybridization. Biochemical analyses also showed that transgenic tomato plants have a substantial amount of relative water content and chlorophyll content under salt stress conditions compared to wild type plants. 3.4. Improved salt stress tolerance in transgenic forage legume crops (Soybean, Alfalfa, L. corniculatus) expressing TaNHX2 gene Legumes are the second foremost crops of agricultural importance throughout the world and provide an essential source of proteins for human as well as animal diet. Legume crops play a significant role in fertilizing the soil via atmospheric nitrogen fixation (Hussain et al., 2010). Especially forage legumes are good resources to afford food and protein security (Kulkarni et al., 2018). However, the cultivation of 4
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Fig. 3. Alignment analysis of wheat NHX proteins (TaNHX1, TaNHX2, and TaNHX3). The conserved amiloride-binding domain is boxed (86–96 amino acid residues).
Fig.4. A flowchart depicting salt tolerance mechanisms in transgenic plants expressing wheat NHX genes.
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candidate genes intricated in response to salinity stress in crop plants would assist plant scientists to accelerate genetic improvement of alfalfa plants through genetic engineering. Zhang et al. (2012) published a report on transgenic alfalfa plants tolerant to salinity stress by introducing TaNHX2 gene through Agrobacterium-mediated genetic transformation. Integration and expression of TaNHX2 gene in T2 transgenic plants is confirmed by RT-PCR and southern hybridization. The transgenic plants have displayed lower electrical conductivity and higher osmotic potential when compared to wild type plants. Interestingly, the vacuolar Na+/H+ antiport activities of transgenic plants were higher under 200 mM NaCl stress and it was not detectable in wild type plants. Their results clearly demonstrated that TaNHX2 as a suitable candidate gene to improve salt stress tolerance in alfalfa plants. L. corniculatus is one of the most important forage leafy legumes with high nutritive value and developing transgenic L. corniculatus plants tolerant to salinity is necessary to sustain the food security (Wang et al., 2018). Jian et al. (2009) validated the function of TaNHX2 gene under salt stress condition in transgenic L. corniculatus plants. The transgenic L. corniculatus plants expressing TaNHX2 have shown greater tolerance against 150 mM NaCl stress. Southern hybridization analysis confirmed the integration of TaNHX2 gene in transgenic plants. This work also suggested that TaNHX2 gene play an important regulatory role in conferring salt tolerance in transgenic L. corniculatus plants.
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Overexpression of wheat Na+/H+ antiporter TNHX1 and Hþ-pyrophosphatase TVP1 improve saltand drought-stress tolerance in Arabidopsis thaliana plants. J. Exp. Bot. 58, 301e–308. Bulle, M., Yarra, R., Abbagani, S., 2016. Enhanced salinity stress tolerance in transgenic chilli pepper (Capsicum annuum L.) plants overexpressing the wheat antiporter (TaNHX2) gene. Mol. Breed. 36, 36. Cao, D., Hou, W., Liu, W., Yao, W.W., Wu, C., Liu, X., Han, T., 2011. Overexpression of TaNHX2 enhances salt tolerance of ‘composite ‘and whole transgenic soybean plants. Plant Cell Tissue Organ Cult. 107, 541–552. Dalal, M., Dani, R.G., Kumar, P.A., 2006. Current trends in the genetic engineering of vegetable crops. Scientia. Hort. 107, 215–225. Dhankher, O.P., Foyer, C.H., 2018. Climate-resilient crops for improving global food security and safety. Plant Cell Environ. 41, 877–884. https://doi.org/10.1111/pce. 13207. Horie, T., Schroeder, J.I., 2004. Sodium transporters in plants. Diverse genes and physiological functions. Plant Physiol. 136, 2457–2462. Hussain, N., Sarwar, G., Schmeisky, H., Al-Rawahy, S., Ahmad, M., 2010. Salinity and drought management in legume crops. In: Yadav, S., Redden, R. (Eds.), Climate Change and Management of Cool Season Grain Legume Crops. Springer, Dordrecht. Jia, Q., Zheng, C., Sun, S., Amjad, H., Liang, K., Lin, W., 2018. The role of plant cation/ proton antiporter gene family in salt tolerance. Biol. Plant. 62 (617), 2018. https:// doi.org/10.1007/s10535-018-0801-8. Jian, B., Hou, W., Wu, C., Liu, B., Liu, W., Song, S., Bi, Y., Han, T., 2009. Agrobacterium rhizogenes-mediated transformation of Superroot-derived Lotus corniculatus plants a valuable tool for functional genomics. BMC Plant Biol. 9, 78. Kulkarni, K.P., Tayade, R., Asekova, S., Song, J.T., Shannon, J.G., Lee, J.-D., 2018. Harnessing the potential of forage legumes, alfalfa, soybean, and cowpea for sustainable agriculture and global food security. Front. Plant Sci. 9, 1314. https://doi. org/10.3389/fpls.2018.01314. Lu, W., Guo, C., Li, X., Duan, W., Ma, C., Zhao, M., Gu, J., Du, X., Liu, Z., Xiao, K., 2014. Overexpression of TaNHX3, a vacuolar Na+/H+ antiporter gene in wheat, enhances salt stress tolerance in tobacco by improving related physiological processes. Plant Physiol. Biochem. 76, 17–28. Munns, R., 2005. Genes and salt tolerance: bringing them together. New Phytol. 167, 645–663. Munns, R., Tester, M., 2008. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59, 651–681. Qadir, M., Quillerou, E., Nangia, V., Murtaza, G., Singh, M., Thomas, R.J., Crechsel, P., Noble, A.D., 2014. Economics of Salt-Induced Land Degradation and Restoration. National Resources Forum, United Nations. https://doi.org/10.1111/1477-8947. 12054. Rahman, M.A., Alam, I., Kim, Y.-G., Ahn, N.-Y., et al., 2015. Screening for salt-responsive proteins in two contrasting alfalfa cultivars using a comparative proteome approach. Plant Physiol. Biochem. 89, 112–122. Shu, K., Qi, Y., Chen, F., Meng, Y., Luo, X., Shuai, H., Zhou, W., Ding, J., Du, J., Liu, J., Yang, F., Wang, Q., Liu, W., Yong, T., Wang, X., Feng, Y., Yang, W., 2017. Salt stress represses soybean seed germination by negatively regulating GA biosynthesis while positively mediating ABA biosynthesis. Front. Plant Sci. 8, 1372. Wang, D., Luo, W., Khurshid, M., Gao, L., Sun, Z., Zhou, M., Wu, Y., 2018. Co-expression of PeDREB2a and KcERF improves drought and salt tolerance in transgenic Lotus corniculatus. J. Plant Growth Regul. 37, 550. Yarra, R., Kirti, P.B., 2019. Expressing class I wheat NHX (TaNHX2) gene in eggplant improves plant performance under saline condition. Funct. Integr. Genom. https:// doi.org/10.1007/s10142-019-00656-5. Yarra, R., He, S.J., Abbagani, S., Ma, B., Bulle, M., Zhang, W.K., 2012. Overexpression of a wheat Na+/H+ antiporter gene (TaNHX2) enhances tolerance to salt stress in transgenic tomato plants (Solanum lycopersicum L.). Plant Cell Tissue Organ Cult. 111 (1), 49–57. Yu, J.N., Huang, J., Wang, Z.N., Zhang, J.S., Chen, S.Y., 2007. An Na+/H+ antiporter gene from wheat plays an important role in stress tolerance. J. Biosci. 32, 1153e–1161. Zhang, Y.M., Liu, Z.H., Wen, Z.Y., Zhang, H.M., Yang, F., Guo, X.L., 2012. The vacuolar Na+/H+ antiport gene TaNHX2 confers salt tolerance on transgenic alfalfa (Medicago sativa). Funct. Plant Biol. 39 (8), 708–716. Zhuang, J., Zhang, J., Hou, X.L., Wang, F., Xiong, A.S., 2014. Transcriptomic, proteomic, metabolomic and functional genomic approaches for the study of abiotic stress in vegetable crops. Crit. Rev. Plant Sci. 33, 225–237.
4. Conclusion and future prospects In recent years, the major focus on plant genetic engineering to develop salt-tolerant crops has been on the expression of vacuolar Na+/ H+ antiporter genes (NHX). Especially, production of transgenic plants (Arabidopsis, tobacco, vegetables and forage legumes) tolerant to salinity stress evidenced the potentiality of wheat NHX genes. The enhanced salinity stress tolerance in above described transgenic plants resulted in the normal growth performance without any defects under salt stress conditions. It has shown the fruitful path for yield increment of vegetable and forage legume crops under saline-prone areas. The expression of TaNHX1, TaNHX2, and TaNHX3 in transgenic plants greatly improved the antioxidant enzyme activities, ion homeostasis, ROS scavenging capacity, osmolyte synthesis with unhampered photosynthetic machinery indicating the regulatory role of these genes under stressful conditions for protecting plants. Acknowledgements The author would like to acknowledge Chinese Academy of Sciences, China and Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Govt. of India for financial support to conduct above described research in various phases. The author declares that this review was prepared without any financial support that might be summed up as a potential conflict of interest. References Ahanger, M.A., Akram, N.A., Ashraf, M., Alyemeni, M.N., Wijaya, L., Ahmad, P., 2017. Plant responses to environmental stresses—from gene to biotechnology. AoB. Plants 9, plx025. https://doi.org/10.1093/aobpla/plx025. Apse, M.P., Aharon, G.S., Snedden, W.S., Blumwald, E., 1999. Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285, 1256–1258. Apse, M.P., Sottosanto, J.B., Blumwald, E., 2003. Vacuolar cation/H+ exchange, ion homeostasis, and leaf development are altered in a T-DNA insertional mutant of AtNHX1, the Arabidopsis vacuolar Na+/H+ antiporter. Plant J. 36, 229–239. Arzani, A., Ashraf, 2016. Smart engineering of genetic resources for enhanced salinity tolerance in crop plants. Crit. Rev. Plant Sci. 35, 146–189. Ashraf, M., Foolad, M.R., 2013. Crop breeding for salt tolerance in the era of molecular
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Contents lists available at ScienceDirect
Plant Gene journal homepage: www.elsevier.com/locate/plantgene
The wheat NHX gene family: Potential role in improving salinity stress tolerance of plants
T
Rajesh Yarra State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
A R T I C LE I N FO
A B S T R A C T
Keywords: NHX antiporters Salinity stress Vegetable crops Forage legumes
Abiotic stress such as salinity adversely influences the growth, development and yield productivity of crop plants. Development of transgenic crops resilient to salinity stress is indispensable to ensure food security around the globe. Identification and functional validation of novel genes during abiotic stress adaptation of plants will afford the basis for effective genetic engineering approaches to enhance salinity stress tolerance of crop plants. The membrane and vacuolar Na+/H+ antiporters provide the best mechanism for ionic homeostasis in plants under salt stress. The function of vacuolar NHX antiporters in plants has been characterized and expressed in heterologous systems to enhance salinity stress tolerance. To date, a total of three vacuolar localized NHX genes have been identified from the wheat genome. Among the three wheat vacuolar NHX antiporters, TaNHX2 plays a vital role in conferring salinity tolerance in various crop plants. In this review, the author has discussed the potential role of wheat NHX genes in conferring salinity tolerance in Arabidopsis, tobacco, vegetable, and legume forage crops via genetic engineering approach. Finally, proposed the future prospects to engineer the higher plants genome with wheat NHX genes for sustainable food production in salinity affected areas.
1. Introduction The extent of agricultural farming area affected by high salinity is increasing day by day around the globe and about 20% of irrigated arable land is already under threaten (Qadir et al., 2014; Munns and Tester, 2008). Salinity stress is one of the most threatening environmental constraints that negatively affects growth, development and overall productivity of plants through induction of ion toxicity, water uptake deficiency, hormonal disturbance and oxidative stress (Ahanger et al., 2017; Ashraf and Foolad, 2013; Munns, 2005). To cope with the high salinity induced effects, plants trigger several stress tolerance responses including compartmentation, exclusion, and secretion of Na+/ H+ ions into vacuoles (Ahanger et al., 2017; Arzani, 2016). The unique structural feature of plant cells is the presence of vacuoles. Plant Na+/ H+ exchangers (NHXs) are intracellular membrane proteins that play a major role in pH, K+ and Na+ homeostasis of cells in higher plants. Most of the Na+/H+ antiporters are localized in the vacuole and involved in sequestration of Na+ within the vacuole under stressful conditions (Jia et al., 2018; Apse et al., 1999, 2003). These vacuolar antiporters regulate the exchange of H+/Na+ across tonoplast to reduce the toxicity of Na+ concentration in the cytoplasm (Arzani, 2016). The vacuolar Na+/H+ antiporters mediate the sequestration of excessive Na+ under salt stress (Blumwald and Poole, 1985, 1987) and
facilitate Na+ uptake into vacuoles, which is driven by the vacuolar proton gradient established by the vacuolar (V-type) proton ATPase that acidifies the vacuolar lumen (Fig. 1). The subsequent vacuolar Na+ sequestration protects important enzymatic reactions in the cytoplasm from excess Na+ levels while maintaining turgor (Horie and Schroeder, 2004). Among the identified NHX genes in higher plants, the wheat NHX genes (TaNHX1, TaNHX2 & TaNHX3) have a prospective role in improving the salt tolerance of various crop plants (Table 1). These three wheat NHX genes were firstly isolated by screening the wheat cDNA library (Brini et al., 2005; Lu et al., 2014; Yu et al., 2007). Under the saline condition, the expression of TaNHX2 and TaNHX3 transcripts is higher in leaves and roots of wheat plants (Lu et al., 2014; Yu et al., 2007) where TaNHX1 expression is higher only in roots compared to leaves (Brini et al., 2005). All these three wheat NHX proteins contain 12 membrane-spanning domains (Fig. 2) and one highly conserved amiloride binding domain (LFFIYLLPPI motif; amino acid residue 86–96) (Fig. 3). These transmembrane domains of wheat NHX proteins are highly conserved with regard to other NHX proteins of plants and responsible for targeting them to tonoplast. The transgenic technology is an important approach for developing salt-tolerant crops to withstand adverse environmental conditions (Dhankher and Foyer, 2018). The crucial step before proceeding with transgenic crops is the identification of potential candidate genes
E-mail address: rajeshyarra@rediffmail.com. https://doi.org/10.1016/j.plgene.2019.100178 Received 18 January 2019; Received in revised form 12 March 2019; Accepted 13 March 2019 Available online 14 March 2019 2352-4073/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Schematic representation showing Na+/H+ exchange across tonoplast and plasma membrane. Excess Na+ is secreted into the vacuole through NHXs (NHX1/NHX2/NHX3). The proton gradient across the vacuolar membrane is established by H+pyrophosphatase (AVP1) and the vacuolar H+ATPase (V- ATPase). The proton gradient across the plasma membrane is established through PMATPase.
yeast transformants expressing TaNHX1 complement yeast nhx1 sensitivity to cationic antibiotic hygromycin (Brini et al., 2005). The yeast transformants expressing TaNHX2, TaNHX3 showed better growth and survival rate compared to control under 400 mM NaCl and 200 mM NaCl stress conditions respectively. These results revealed that wheat NHX genes can complement the yeast mutant deficient in vacuolar Na+/H+ antiporter exchanging activities for better survival under salt stress conditions.
serving as key regulators of different metabolic pathways, including osmolyte synthesis, ion homeostasis through selective ion uptake (Ahanger et al., 2017). There are various NHX genes that might be utilized to transform crops to enhance salinity tolerance. In this review, I focus on the three important wheat NHX genes that greatly improve salinity tolerance in various plants. A complement analysis of wheat NHX genes in nhx yeast mutants confirmed their role under salt stress conditions (Brini et al., 2005; Lu et al., 2014; Yu et al., 2007). Further studies revealed that the ectopic expression of TaNHX1 and TaNHX3 in tobacco improves the growth performance under saline conditions (Brini et al., 2007; Lu et al., 2014) (Table 1). Till now, the effect of TaNHX1 and TaNHX3 expression is only reported in transgenic tobacco plants under salt stress conditions (Table 1). But functional validation of TaNHX2 expression is highly explored in transgenic vegetable plants and forage legume crops (Table 1). Especially, our research groups have been working in China and India since 2011 in this research area and successfully generated transgenic vegetable plants (Eggplant, Chill pepper and Tomato) tolerant to high salinity stress with TaNHX2 gene through genetic engineering approach. In this review, I summarize the role of these potential candidate genes (TaNHX1, TaNHX2 & TaNHX3) for improving the salt tolerance in various plants with special emphasis on major vegetable plants (Eggplant, Chilli pepper, and Tomato) and forage legume crops (soybean, alfalfa, and L. corniculatus).
3. Enhanced salt tolerance in transgenic plants expressing wheat NHX genes 3.1. TaNHX1 improves salt tolerance in A. thaliana plants Brini et al. (2005) reported that expression of TaNHX1 in Arabidopsis plants significantly improves the salinity tolerance of transgenic plants under salt stress conditions (200 mM NaCl). The Arabidopsis transgenic plants were generated by Agrobacterium-mediated transformation using binary vector pCB302.2 that contains TaNHX1 and TVP1 with a tandem repeat of the 35S promoter, the 35S terminator, and the BAR gene for resistance between the NOS promoter and terminator. The data have shown that the greater accumulation of Na+/K+ contents in the leaves of transgenic Arabidopsis plants compared to wild type plants under salt stress conditions. Moreover, TaNHX1 expressing Arabidopsis plants also displayed greater stability of relative water content compared with wild type plants under salt stress conditions. Taken together, all these results clearly indicate that TaNHX1 plays an important role in improving the salinity stress tolerance of transgenic plants under high NaCl conditions.
2. Complement analysis of wheat NHX genes in yeast transformants The expression of wheat NHX genes namely, TaNHX1, TaNHX2, and TaNHX3 in salt-sensitive yeast mutants (nhx1: W303) improved the survival rate of yeast mutants under salt stress conditions. The pRS8 plasmid harboring TaNHX1 under the control PMA1 promoter was used to generate yeast transformants (Brini et al., 2005). A yeast expression vectors pYES2 harboring TaNHX2 (pYES2-TaNHX2) (Yu et al., 2007) and TaNHX3 (pYES2-TaNHX3) (Lu et al., 2014) under the control of GAL1 promoter, were used to generate yeast transformants. These binary cassette plasmids and the control plasmids (pRS8, pYES2) were subjected to transform nhx1: W303, a yeast strain with a deficient function in the vacuolar Na+/H+ antiporter exchanging activity. The
3.2. TaNHX3 improves salt tolerance in tobacco plants Lu et al. (2014) generated the transgenic tobacco plants with enhanced salinity tolerance via overexpressing TaNHX3 gene. The binary plasmid pCAMBIA3301 containing TaNHX3 gene was used to generate transgenic tobacco plants through Agrobacterium-mediated transformation. The salt tolerance and stress associated physiological processes were significantly improved in transgenic tobacco plants. Transgenic plants accumulated more Na+ contents, higher fresh and dry weights, 2
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Zhang et al. (2012) Jian et al. (2009) Lu et al. (2014) Transgenic alfalfa plants were tolerant to 200 mM NaCl stress Transgenic plants were successfully grown in 150 mM NaCl stress Transgenic tobacco plants were tolerant to 150 mM NaCl stress Alfalfa (cv.Sandeli) L. corniculatus Tobacco (cv.Wisconsin38) TaNHX2 TaNHX2 TaNHX3
Brini et al. (2007) Yarra and Kirti (2019) Bulle et al. (2016) Yarra et al. (2012) Cao et al. (2011)
Floral buds Leaves Cotyledons Cotyledons Roots, Cotyledons Cotyledonary nodes Cotyledons Roots Leaves
Arabidopsis thaliana (eco:col) Eggplant (cv.PPL) Chilli Pepper (cv.G4) Tomato (cv. PED) Soyabean (cv.Zigongdongdou)
Transgenic plants grew well under 200 mM NaCl stress Transgenic eggplants exhibited an increased tolerance levels to 200 mM NaCl stress Transgenic Cilli Pepper plants were found to be tolerant against 200 mM NaCl stress Improved the salinity tolerance in transgenic tomato plants against 150 mM NaCl stress Higher level of salt tolerance was found in the transgenic lines against 200 mM and 150 mM NaCl stress
Vegetables play a vital role in human nutrition as they are rich sources for minerals, micronutrients, vitamins, antioxidants, phytosterols, and dietary fiber. Vegetable crop cultivation is also an imperative need to boost the agricultural economy for many countries (Dalal et al., 2006). The solanaceae or nightshade family of vegetables includes tomato, eggplant, and chilli pepper are indispensable for the human diet because of their nutritional value. Salinity stress causes considerable loss of these important vegetable crops productivity and quality in abiotic stress-prone areas (Zhuang et al., 2014; Bulle et al., 2016; Bai et al., 2018). Development of transgenic vegetable crops with an inherent capacity to withstand salinity stress would help in alleviating the vegetable production for sustainable food production and security. Selection of a suitable candidate gene is an important strategy to develop salt-tolerant transgenic plants. In hunt of a suitable candidate gene, we have chosen wheat TaNHX2 gene to develop the transgenic eggplant, chilli pepper, and tomato plants tolerant to salinity stress. Since 2011, we have been trying to achieve the goal and finally produced the salt tolerant eggplant, chilli pepper, and tomato plants through genetic engineering approach (Yarra and Kirti, 2019; Bulle et al., 2016; Yarra et al., 2012). These reports proved the potentiality of TaNHX2 gene for improving the salt tolerance of transgenic vegetable plants. We have used transformation vector pBIN438 harboring TaNHX2 gene, driven by the double 35S promoter and NPTII as a selection marker for the transformation of vegetable plants. Recently, we have published a report on the successful production of transgenic eggplants tolerant to salt stress (200 mM NaCl) by introducing TaNHX2 gene in eggplant (cv.PPL) genome via Agrobacteriummediated genetic transformation (Yarra and Kirti, 2019). Integration and expression of TaNHX2 transcripts in transgenic eggplants were confirmed by RT-PCR, qRT-PCR and southern hybridization. Transgenic eggplants (T2) expressing TaNHX2 displayed improved growth performance, stable leaf relative water content and chlorophyll content, proline accumulation, improved photosynthetic efficiency, transpiration rate, stomatal conductivity than the non-transformed plants under salinity stress (200 mM NaCl). The eggplant transgenics also showed decreased MDA content, hydrogen peroxide, oxygen radical production accompanying with the significant increase of antioxidant enzymes (SOD, APX, GPX, and GR) activity than non-transformed plants under salt stress (200 mM NaCl). The adaptation of plants to salinity stress is determined by biochemical alternations that enable the retention of water content, maintenance of ion homeostasis and photosynthetic apparatus protection (Dhankher and Foyer, 2018). The free radical scavenging capacity of the plants under salinity stress is an essential need. TaNHX2 expression in eggplants also triggered the biochemical pathways such as ion homeostasis, radical scavenging capacity and increased level of antioxidant enzymes for the survival under salinity conditions without any growth defects (Fig. 4). Bulle et al. (2016) published a report on transgenic chill pepper plants tolerant to salinity stress. We have successfully overcome the various barriers to achieve the genetic transformation of chilli pepper (cv.G4) plants via the introduction of TaNHX2 gene through Agrobacterium-mediated genetic transformation. Integration and expression of the TaNHX2 gene in chilli pepper plants genome is confirmed by RTPCR and southern hybridization. T1 transgenic plants exhibited better
tumefaciens tumefaciens (LBA4404) tumefaciens (LBA4404) tumefaciens (LBA4404) rhizogenes (K599) tumefaciens (EHA101) tumefaciens (EHA105) rhizogenes (K599) tumefaciens (EHA105) Agrobacterium Agrobacterium Agrobacterium Agrobacterium Agrobacterium Agrobacterium Agrobacterium Agrobacterium Agrobacterium
TaNHX1 TaNHX2 TaNHX2 TaNHX2 TaNHX2
References Character/trait improved Plant Explants
Gene
more accumulative nitrogen, phosphorus and potassium, higher contents of chlorophyll, carotenoid, soluble protein. Transgenic tobacco plants also displayed a significant increase of the antioxidant enzyme activities including superoxide dismutase, catalase, and peroxidase associated with the lower amounts of malondialdehyde and H2O2 compared to wild type plants. These results clearly demonstrated that TaNHX3 also play an important regulatory role in ROS scavenging capacity under salt stress conditions (Fig. 4). 3.3. TaNHX2 improves salt tolerance in transgenic vegetable plants (eggplant, chilli pepper, tomato)
Method of transformation
Table 1 Salt tolerant transgenic plants developed so far by introducing wheat NHX genes.
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Fig. 2. Diagram showing the twelve conserved transmembrane domains of wheat NHX proteins (TaNHX1, TaNHX2, and TaNHX3).
forage legume crops is confronting with adverse environmental conditions including salinity stress, indicating the need for tolerant varieties that can withstand and acclimatize to the deleterious effects of salinity stress. Advancements in genetic engineering facilitated to develop salinity tolerant varieties by introducing heterologous salt responsive candidate genes. In view of this, researchers introduced wheat NHX gene in some forage legume crops and developed salt-tolerant transgenic crops, which includes soybean, alfalfa, L. corniculatus (Cao et al., 2011; Zhang et al., 2012; Jian et al., 2009). Soybean (Glycine max (L.) Merrill) is an important plant source for the human diet as a protein resource and also used as an oil crop throughout the world. Salinity stress has adverse effects on seed germination, vegetative and reproductive growth of soybean plants (Shu et al., 2017). The improvement of soybean by genetic engineering is needed to withstand the salinity stress damage on soybean plants. Cao et al. (2011) reported the transformation of soybean plants (cv. Zigongdongdou) with TaNHX2 gene using the Agrobacterium-mediated transformation method. They have performed hairy root transformation as well as whole plant transformation via A. rhizogenes and A. tumefaciens using the binary vectors pGUS-TaNHX2, pTF101.1-TaNHX2 respectively. Interestingly, overexpression of TaNHX2 in soybean enhanced salt tolerance of transgenic hairy roots as well as composite and whole transgenic plants against 150 mM NaCl stress conditions. Alfalfa (Medicago sativa L.) is a crucial global perennial leguminous forage crop in temperate regions. Alfalfa crop productivity is adversely affected by salinity present in the soil (Rahman et al., 2015). Dissecting
growth performance under 200 mM NaCl conditions compared to wild type plants. Biochemical parameters assayed to analyze the salt tolerance capacity of transgenic plants under stressful conditions. Transgenic plants expressing TaNHX2 displayed increased levels of chlorophyll content, relative water content, superoxide dismutase, ascorbate peroxidase and reduced levels of hydrogen peroxide, malondialdehyde contents compared to wild type plants. Yarra et al. (2012) reported the successful transformation of tomato (cv.PED) plants with TaNHX2 gene through Agrobacterium-mediated approach. The transgenic tomato plants (T1) showed enhanced growth performance under 150 mM NaCl stress conditions compared to wild type plants. Integration and expression of TaNHX2 gene in the tomato genome is confirmed by RT-PCR and southern hybridization. Biochemical analyses also showed that transgenic tomato plants have a substantial amount of relative water content and chlorophyll content under salt stress conditions compared to wild type plants. 3.4. Improved salt stress tolerance in transgenic forage legume crops (Soybean, Alfalfa, L. corniculatus) expressing TaNHX2 gene Legumes are the second foremost crops of agricultural importance throughout the world and provide an essential source of proteins for human as well as animal diet. Legume crops play a significant role in fertilizing the soil via atmospheric nitrogen fixation (Hussain et al., 2010). Especially forage legumes are good resources to afford food and protein security (Kulkarni et al., 2018). However, the cultivation of 4
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Fig. 3. Alignment analysis of wheat NHX proteins (TaNHX1, TaNHX2, and TaNHX3). The conserved amiloride-binding domain is boxed (86–96 amino acid residues).
Fig.4. A flowchart depicting salt tolerance mechanisms in transgenic plants expressing wheat NHX genes.
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candidate genes intricated in response to salinity stress in crop plants would assist plant scientists to accelerate genetic improvement of alfalfa plants through genetic engineering. Zhang et al. (2012) published a report on transgenic alfalfa plants tolerant to salinity stress by introducing TaNHX2 gene through Agrobacterium-mediated genetic transformation. Integration and expression of TaNHX2 gene in T2 transgenic plants is confirmed by RT-PCR and southern hybridization. The transgenic plants have displayed lower electrical conductivity and higher osmotic potential when compared to wild type plants. Interestingly, the vacuolar Na+/H+ antiport activities of transgenic plants were higher under 200 mM NaCl stress and it was not detectable in wild type plants. Their results clearly demonstrated that TaNHX2 as a suitable candidate gene to improve salt stress tolerance in alfalfa plants. L. corniculatus is one of the most important forage leafy legumes with high nutritive value and developing transgenic L. corniculatus plants tolerant to salinity is necessary to sustain the food security (Wang et al., 2018). Jian et al. (2009) validated the function of TaNHX2 gene under salt stress condition in transgenic L. corniculatus plants. The transgenic L. corniculatus plants expressing TaNHX2 have shown greater tolerance against 150 mM NaCl stress. Southern hybridization analysis confirmed the integration of TaNHX2 gene in transgenic plants. This work also suggested that TaNHX2 gene play an important regulatory role in conferring salt tolerance in transgenic L. corniculatus plants.
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Overexpression of wheat Na+/H+ antiporter TNHX1 and Hþ-pyrophosphatase TVP1 improve saltand drought-stress tolerance in Arabidopsis thaliana plants. J. Exp. Bot. 58, 301e–308. Bulle, M., Yarra, R., Abbagani, S., 2016. Enhanced salinity stress tolerance in transgenic chilli pepper (Capsicum annuum L.) plants overexpressing the wheat antiporter (TaNHX2) gene. Mol. Breed. 36, 36. Cao, D., Hou, W., Liu, W., Yao, W.W., Wu, C., Liu, X., Han, T., 2011. Overexpression of TaNHX2 enhances salt tolerance of ‘composite ‘and whole transgenic soybean plants. Plant Cell Tissue Organ Cult. 107, 541–552. Dalal, M., Dani, R.G., Kumar, P.A., 2006. Current trends in the genetic engineering of vegetable crops. Scientia. Hort. 107, 215–225. Dhankher, O.P., Foyer, C.H., 2018. Climate-resilient crops for improving global food security and safety. Plant Cell Environ. 41, 877–884. https://doi.org/10.1111/pce. 13207. Horie, T., Schroeder, J.I., 2004. Sodium transporters in plants. 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Plant Sci. 9, 1314. https://doi. org/10.3389/fpls.2018.01314. Lu, W., Guo, C., Li, X., Duan, W., Ma, C., Zhao, M., Gu, J., Du, X., Liu, Z., Xiao, K., 2014. Overexpression of TaNHX3, a vacuolar Na+/H+ antiporter gene in wheat, enhances salt stress tolerance in tobacco by improving related physiological processes. Plant Physiol. Biochem. 76, 17–28. Munns, R., 2005. Genes and salt tolerance: bringing them together. New Phytol. 167, 645–663. Munns, R., Tester, M., 2008. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59, 651–681. Qadir, M., Quillerou, E., Nangia, V., Murtaza, G., Singh, M., Thomas, R.J., Crechsel, P., Noble, A.D., 2014. Economics of Salt-Induced Land Degradation and Restoration. National Resources Forum, United Nations. https://doi.org/10.1111/1477-8947. 12054. Rahman, M.A., Alam, I., Kim, Y.-G., Ahn, N.-Y., et al., 2015. Screening for salt-responsive proteins in two contrasting alfalfa cultivars using a comparative proteome approach. Plant Physiol. 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4. Conclusion and future prospects In recent years, the major focus on plant genetic engineering to develop salt-tolerant crops has been on the expression of vacuolar Na+/ H+ antiporter genes (NHX). Especially, production of transgenic plants (Arabidopsis, tobacco, vegetables and forage legumes) tolerant to salinity stress evidenced the potentiality of wheat NHX genes. The enhanced salinity stress tolerance in above described transgenic plants resulted in the normal growth performance without any defects under salt stress conditions. It has shown the fruitful path for yield increment of vegetable and forage legume crops under saline-prone areas. The expression of TaNHX1, TaNHX2, and TaNHX3 in transgenic plants greatly improved the antioxidant enzyme activities, ion homeostasis, ROS scavenging capacity, osmolyte synthesis with unhampered photosynthetic machinery indicating the regulatory role of these genes under stressful conditions for protecting plants. Acknowledgements The author would like to acknowledge Chinese Academy of Sciences, China and Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Govt. of India for financial support to conduct above described research in various phases. The author declares that this review was prepared without any financial support that might be summed up as a potential conflict of interest. References Ahanger, M.A., Akram, N.A., Ashraf, M., Alyemeni, M.N., Wijaya, L., Ahmad, P., 2017. Plant responses to environmental stresses—from gene to biotechnology. AoB. Plants 9, plx025. https://doi.org/10.1093/aobpla/plx025. Apse, M.P., Aharon, G.S., Snedden, W.S., Blumwald, E., 1999. Salt tolerance conferred by overexpression of a vacuolar Na+/H+ antiport in Arabidopsis. Science 285, 1256–1258. Apse, M.P., Sottosanto, J.B., Blumwald, E., 2003. Vacuolar cation/H+ exchange, ion homeostasis, and leaf development are altered in a T-DNA insertional mutant of AtNHX1, the Arabidopsis vacuolar Na+/H+ antiporter. Plant J. 36, 229–239. Arzani, A., Ashraf, 2016. Smart engineering of genetic resources for enhanced salinity tolerance in crop plants. Crit. Rev. Plant Sci. 35, 146–189. Ashraf, M., Foolad, M.R., 2013. Crop breeding for salt tolerance in the era of molecular
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