Halophytes: Potential Resources for Salt Stress Tolerance Genes and Promoters

Halophytes have demonstrated their capability to thrive under extremely saline conditions and thus considered as one of the best germplasm for saline agriculture. Salinity is a worldwide problem, and the salt-affected areas are increasing day-by-day because of scanty rainfall, poor irrigation system, salt ingression, water contamination, and other environmental factors. The salinity stress tolerance mechanism is a very complex phenomenon, and some pathways are coordinately linked for imparting salinity tolerance. Though a number of salt responsive genes have been reported from the halophytes, there is always a quest for promising stress-responsive genes that can modulate plant physiology according to the salt stress. Halophytes such as Aeluropus, Mesembryanthemum, Suaeda, Atriplex, Thellungiella, Cakile, and Salicornia serve as a potential candidate for the salt-responsive genes and promoters. Several known genes like antiporters (NHX, SOS, HKT, VTPase), ion channels (Cl−, Ca2+, aquaporins), antioxidant encoding genes (APX, CAT, GST, BADH, SOD) and some novel genes such as USP, SDR1, SRP etc. were isolated from halophytes and explored for developing stress tolerance in the crop plants (glycophytes). It is evidenced that stress triggers salt sensors that lead to the activation of stress tolerance mechanisms which involve multiple signaling proteins, up- or down-regulation of several genes, and finally the distinctive or collective effects of stress-responsive genes. In this review, halophytes are discussed as an excellent platform for salt responsive genes which can be utilized for developing salinity tolerance in crop plants through genetic engineering.


INTRODUCTION
Salinization is a worldwide problem in which salts gradually accumulate in the soil. In this process, water-soluble salts are deposited in the soil to an extent that affects crop productivity, microbial community, and agricultural economics (FAO, 2016). The salinization eventually transforms a fertile land to barren. The process destroys all vegetation and other organisms living in the soil and thus it is detrimental to the environmental health. Most of the world's land is not cultivated, and over 2% of the total land is affected by salinity (FAO; Land and Plant Nutrition Management Service). A significant proportion of cultivated land is salt-affected, and out of the current 230 million ha of irrigated land, 45 million ha are salt-affected whereas 32 million are salt-affected to varying degrees (FAO, 2008).
Halophytes are salt-resistant or salt-tolerant plants and have remarkable ability to complete their life cycle in saline condition. During evolution, they have developed different morphological, anatomical, and physiological strategies to proliferate in highsalt environments Grigore et al., 2014). Halophytes have occasionally been reviewed for their general physiology (Flowers, 1985), ecophysiology (Ball, 1988), photosynthesis (Rozema and Van Diggelen, 1991;Lovelock and Ball, 2002), response to oxidative stress (Jithesh et al., 2006), flooding tolerance , salinity tolerance , and adaptations . Additionally, other researchers have also examined halophytes under special topics as sustainable cultivation, saline agriculture, and integrative anatomy Grigore et al., 2014;Xu et al., 2016).
Halophytes that consistently require salt for their growth are referred to "obligate halophytes" (Braun-Blanquet, 1932), but some halophytes have the ability to grow on the soil devoid of salt are called as "facultative halophytes" (Polunin, 1960). Halophytes are obligate and facultative based upon salt demand and tolerance for sodium salts. Previously, a study has been carried out with some selected halophytes to investigate the salt requirement for growth and development (Grigore et al., 2012). Researchers concluded that salts are not compulsorily required for the development of halophytic species but the availability of water and nutrients are also key limiting factors for growth in natural saline habitats (Grigore et al., 2012). Further, they hypothesized that halophytes are mostly distributed in saline areas to avoid competition with glycophytic species.
Ecophysiological characteristics are used by Cushman (2001) to differentiate between obligate, facultative, and habitat-indifferent halophytes. Habitat-indifferent halophytes are undistinguished to their habitat, usually, prefer to live in a salt-free soil but have the ability to cope with the saline condition (Cushman, 2001). Recently,  proposed a new type of classification of halophytes; extreme-halophyte (irreversible and reversible) and mesohalophytes, by integrating anatomy observations with ecological factors (salinity). Extreme-halophytes are well-adapted extreme halophytes and growing exclusively in saline environments. Furthermore, the habitat of these halophytes may be irreversible or reversible. They concluded that Chenopodiaceae (now included in the family Amaranthaceae) succulent species (Salicornia, Suaeda, Halimione, and Petrosimonia) are extreme halophytes and best adapted to high salinity conditions. Halophytes such as Atriplex, Bassia, and Camphorosma are not strictly related to increased salinity, therefore may be classified as reversible halophytes. There is always a difficulty with the distinct terminology of halophytes because the definition is still obscure and  discussed a short historical evolution of halophytes definition in chronological order.
Advanced and novel stress-tolerant mechanisms are difficult to study with the model plant Arabidopsis as some mechanisms are unique to halophytes. The comparative genomics of Mesembryanthemum crystallinum and Arabidopsis thaliana confirmed that some transcripts present in former and later do not have counterparts (Wang et al., 2004). Some other halophytes, Suaeda species, and Atriplex species have been investigated to unravel molecular mechanism of stress tolerance. Among all, Thellungiella halophila is one of the halophytes emerging as a model halophyte for the study of abiotic stress tolerance mechanism (Wang et al., 2004;Amtmann, 2009). Halophyte Cakile maritima and Suaeda maritima (Megdiche et al., 2009;Sahu and Shaw, 2009) are considered as model plants for the transcript profiling and Salicornia brachiata as a potential halophyte for new and useful salt-tolerant genes Udawat et al., 2016Udawat et al., , 2017. In this review, halophytes are discussed as resources for salt stress tolerance genes, which can be explored further for developing abiotic stress tolerance crops for sustainable agriculture.

Salt Tolerance Mechanism in Halophytes: A Glimpse
Halophytes are well-adapted and thrive under high salinity by using two strategies, salt tolerance, and salt avoidance. Generally, halophytes follow three mechanisms of salt tolerance; reduction of the Na + influx, compartmentalization, and excretion of sodium ions (Flowers andColmer, 2008, 2015). Adaptations involved in salt avoidance are secretion, shedding, and succulence (discussed in Waisel, 1972;Rozema, 1995;Aslam et al., 2011;Shabala et al., 2014). In brief, secretion is a complex mechanism, and salt-secreting structures (salt hairs or salt glands) are distributed in halophytes. Some halophytes are capable of excreting excess salt in the form of a liquid which becomes crystals in contact with air and may visible on the plant leaf surface. In some halophytes, shedding of the old leaves which are grown under high salt concentrations is another strategy to avoid the salt toxicity. Grigore et al. (2014) discussed the different aspects of the various adaptive structures of halophytes in an integrative way at the anatomy level.
The salt tolerance mechanism is coordinately linked (Figure 1) with signal transduction, ROS generation and detoxification pathways, osmoregulation or ion homeostasis through osmoprotectants, and differential expression of salt responsive genes and transcription factors Rajalakshmi and Parida, 2012;Himabindu et al., 2016;Khan et al., 2016;Muchate et al., 2016). ROS detoxification pathways include antioxidative enzymes which play a protective role in scavenging toxic radicals (Das and Strasser, 2013). Salt sequestration into cell vacuoles through transporters is another key mechanism employed by halophytes to maintain a high cytosolic K + /Na + ratio and thus control the salt concentrations in the cytosol (Kronzucker and Britto, 2011;Sreeshan et al., 2014). Accumulation of osmoprotectants such as proline, glycine betaine, polyphenols, soluble sugars, and inorganic ions is a conventional plant defense mechanism routinely used by halophytes to cope with stresses (Lokhande and Suprasanna, 2012;Patel et al., 2016). At the molecular level, halophytes impart salt tolerance by regulating stress-responsive genes through ABA-dependent or ABA-independent regulation mechanism. Overall, salt tolerance in a halophyte is a complex network that involves the interactions of multiple physiological responses directive by several genes and gene products (Figure 1). Overall, FIGURE 1 | A generalized schematic representation of salinity stress tolerance mechanism in a plant.
halophytic salt tolerance defense mechanism includes changes in ion homeostasis (both influx and efflux), the formation of osmoprotectants, activation of crosstalk genes, induction of antioxidants, and the development of salt gland or bladders (Shabala et al., 2014;Slama et al., 2015;Himabindu et al., 2016).

Salt Responsive Genes from Halophytes: An Overview
Halophytes have been studied extensively for their ecological, physiological, anatomical, and biochemical responses toward salinity Aslam et al., 2011;Shabala, 2013;Ventura et al., 2015). Furthermore, halophytes were also explored for saline agriculture and examined as bioenergy crop (Rozema and Schat, 2013;Sharma et al., 2016). However, little information is available on well-defined molecular defense mechanism of halophytes against salt stress (Anjum et al., 2012;Joshi et al., 2015). Surprisingly, a non-tolerant plant, A. thaliana is widely explored as a model plant to investigate the molecular mechanism of salt stress tolerance (Sanders, 2000;Zhu, 2001). Additionally, this plant is also exploited for the gene mining of salt stress-responsive genes for the improvement of tolerance in transgenic crops (Zhu, 2000).
It is a general assumption that halophytes are salt resistant while glycophytes are sensitive, but there are several species considered traditionally as glycophytes are resistant or tolerant to salt and some halophytes may be sensitive to several environmental stresses. Recently, it is experimentally proven that halophytes are one of the most appropriate models for the studying different salt stress tolerance mechanisms (Shabala, 2013;Himabindu et al., 2016). A number of evidences suggest that all plants have almost similar salt tolerance regulatory mechanisms and there are quantitative differences rather than qualitative between halophyte and glycophyte (Anjum et al., 2012;Rai et al., 2012;Bartels and Dinakar, 2013;Sreeshan et al., 2014;Joshi et al., 2015;Volkov, 2015;Muchate et al., 2016). It may be because of higher expression of key genes involved in the salt stress tolerance mechanism, or halophytic proteins are intrinsically more active than the corresponding glycophytic proteins (Anjum et al., 2012;Das and Strasser, 2013;Himabindu et al., 2016;Muchate et al., 2016).
Different genomic and transcriptomics efforts have been made to isolate salt responsive genes from some halophytes followed by their functional validation through transgenic approaches. The overexpression of several halophytic genes, under the control of a non-specific 35SCaMV promoter, have been claimed to enhance abiotic stress tolerance in the glycophytic recipients ( Table 1). A number of crops have been transformed with halophytic genes for the improvement of salt tolerance. Most of these genes encode for Na + /H + antiporters (vacuolar or plasma membrane), vacuolar pyrophosphatase, potassium transporters, ion channels, antioxidants, ROS scavengers, and proteins that involve in protective function and signal transduction. Additionally, some novel salt responsive genes were also cloned and characterized from halophytes like S. brachiata (Udawat et al., 2014(Udawat et al., , 2017Singh et al., 2016).
A close relative of thoroughly explored glycophytic crucifer A. thaliana, Thellungiella salsuginea, which was earlier classified as T. halophila is a halophyte, exhibiting a high tolerance to salt and drought, considered as a potential model for abiotic stress tolerance studies by some researchers (Amtmann, 2009;Bartels and Dinakar, 2013). The genome sequence of T. salsuginea provides evidence about the genetic basis of abiotic stress defense mechanisms, and comparative genomics identified this plant as a gene resource for cation transporters, abscisic acid signaling genes, and other upregulated genes that showing a response to stressful environments (Wu et al., 2012). Furthermore, microarray analysis exhibited that only few genes were induced in Thellungiella compared to Arabidopsis under salt stress (Taji et al., 2004). Another study reveals that about 154 genes were differentially regulated in Thellungiella compared to Arabidopsis under varying stress (Wong et al., 2006).
Similarly, another halophytic relative of the model plant Arabidopsis, Lepidium crassifolium showed salt, osmotic and oxidative stresses tolerance. Random genes were transferred from L. crassifolium to A. thaliana, and it was observed that independent transgenic lines enhanced tolerance under several stress conditions (Rigó et al., 2016). Approximately 15% of functionally unknown genes were additionally expressed under salt stress compared to the non-stress conditions in M. crystallinum (Cushman and Bohnert, 2000;Kore-eda et al., 2004).
An extreme halophyte S. brachiata grows luxuriantly on salt marshes and also frequently encountered with different environmental stresses. Since, S. brachiata has unique opportunity to sustain adverse conditions and thus considered as a rich source of stress responsive genes and promoters Chaturvedi et al., 2012;Singh et al., 2014a;Tiwari et al., 2014Tiwari et al., , 2016Udawat et al., 2016). The salt responsive genes from S. brachiata have been utilized to develop salt stress tolerant transgenic crops such as jatropha, cumin, castor, and peanuts (Joshi et al., 2011;Singh et al., 2014b;Patel et al., 2015;Tiwari et al., 2015a;Jha et al., 2016;Pandey et al., 2016) using different genetic transformation methods (Singh et al., 2010;Joshi et al., 2012;Pandey et al., 2013;Tiwari et al., 2015b). Furthermore, Salicornia also owns unique oligosaccharides , metabolites , sulfur-rich seed-storage proteins  and thus considered as a functional food. Transcriptomics of Porteresia coarctata, a wild relative of rice showing high salinity and submergence tolerance revealed a total of 152,367 unique transcript sequences (Garg et al., 2014). A total of 15,158 genes, involved in salinity and submergence tolerance were identified to unravel key metabolic pathways. These genes can be explored further to understand and engineer salinity and submergence tolerance in rice (Garg et al., 2014).

Promoters of Salt-Responsive Halophytic Genes: At a Glance
A strong and well-regulated promoter is required for the engineering of crop plants to achieve the desired level of expression of a transgene. A comparative transcriptome analysis revealed that many stress-related genes constitutively expressed at higher level in T. halophila compared to their homologs of A. thaliana (Taji et al., 2004(Taji et al., , 2010. This report suggests an efficient transcriptional regulatory network for stress responsive genes in halophytes. Recently, cis-regulatory elements of different stress responsive genes from some halophytes have been studied, and the presence of various stress-inducible motifs was observed (Tiwari et al., , 2016. Yin et al. (2002) found that the promoter of AcBADH gene from Atriplex centralasiatica is strongly induced by salt stress and possesses two salt-responsive enhancer regions (located from −1,115 to −890 and −462 to −230) and one silencer region (located between −890 and −641).
The SlBADH gene promoter fragment (−300 bp only) from Suaeda liaotungensis showed about 6.3-fold expression under salt stress (400 mmol/l NaCl) compared to control (non-stressed) condition . The TsVP1 gene promoter from halophyte T. halophila contained 130 bp specific cis-acting element and showed a higher expression of GUS in transgenic Arabidopsis under salt stress (Sun et al., 2010). Similarly, an 897 bp promoter region of SlPEAMT gene (S. liaotungensis) showed an 18.6-fold increase in the GUS activity under NaCl stress (200 mmol/l) treatment (Li et al., 2016). These results suggest that even a small fragment of promoter can also contain essential cis-acting elements to regulate gene expression under stress. The promoters of CMO genes from S. liaotungensis and Salicornia europaea also possessed basic elements and demonstrated to be salt inducible (Li et al., 2007;Wu et al., 2011). Schaeffer et al. (1995) identified enhancer and silencer regions involved in the transcriptional activation of salt-responsive expression of CAM (Crassulacean Acid Metabolism) genes in the halophyte M. crystallinum.
An age-dependent, abiotic-stress-inducible, organ-specific, and tissue-specific promoter, AlSAP was reported from Aeluropus littoralis (Saad et al., 2011). Furthermore, gusA exhibited same expression level under the control of AlSAP gene promoter in transgenic rice as AlSAP transcript in A. littoralis (Ben- Saad et al., 2015). They also concluded that the regulatory regions of two orthologs AlSAP and OsSAP9 (from rice) have a different specificity of regulation and stress induction in rice. Sun et al. (2010) found a 130 bp specific cis-acting element in the promoter region of vacuolar H + -pyrophosphatase from a halophyte T. halophila (TsVP1) which enhances the expression of GUS in transgenic Arabidopsis under salt stress. The CBL1 gene promoter isolated from Ammopiptanthus mongolicust controlled the expression of the reporter gene under abiotic and biotic stress conditions (Guo et al., 2010). A model, proposed for transcriptional regulation of the SbpAPX gene (from S. brachiata) showed the presence of enhancer and repressor binding sites in the cis-regulatory elements along with stress-inducible motifs . Similarly, the SbGSTU promoter showed the presence of a number of abiotic stress responsive cisregulatory motifs which regulate the expression of GSTU gene in S. brachiata (Tiwari et al., 2016). Therefore, based on different reports, halophytic promoters emerge as a promising candidate for engineering abiotic stress tolerance in crops for high-level expression of transgenes.

Salt Tolerant Genes from Halophytes and Glycophytes: A Comparative Analysis
Among different strategies; Na + efflux, compartmentalization of Na + in vacuoles and prevention of Na + influx are the most common, governed by antiporters and regulated by a multigene family (Rajendran et al., 2009;Kronzucker and Britto, 2011). A number of antiporters isolated from both glycophytes and halophytes are functionally characterized (Kronzucker and Britto, 2011;Sreeshan et al., 2014). The overexpression of glycophytic transporters encoding genes (NHX, SOS, HKT, ATPase, etc.), under the control of nonspecific CaMV35S promoter, showed tolerance in the range of 150-250 mM NaCl, however their halophytic homologs may provide tolerance up to 400 mM NaCl (reviewed in Kronzucker and Britto, 2011;Sreeshan et al., 2014;Volkov, 2015). In several previous studies, the effects of overexpression of halophytic genes were commonly observed under salt stress treatments, however, negligible differences were observed between wildtype plants and the transgenic lines under control (unstressed) conditions Joshi et al., 2012;Volkov, 2015;Tiwari et al., 2015a;Singh et al., 2016;Udawat et al., 2016Udawat et al., , 2017.
Similar to NHX gene family, the overexpression of other halophytic genes such as SbpAPX, SbUSP, and SbGSTU showed better salinity tolerance (200-300 mM NaCl) in the transgenic plants compared to their glycophytic homologs Singh et al., 2014a,b;Udawat et al., 2016). The transgenic Arabidopsis plants, overexpressing the TIP1 gene from the halophyte T. salsuginea exhibited better salt tolerance compared to the same gene from glycophyte Panax ginseng (Peng et al., 2007). Similarly, APX and GST from rice showed lower tolerance up to 150-200 mM compared to the same genes (200-300 mM NaCl) from halophyte S. brachiata in the transgenic plants (Lu et al., 2007;Jha et al., 2011;Sharma et al., 2014;Singh et al., 2014a). Recently, it was reported that over-expression of a stress-associated protein gene (AlSAP) from A. littoralis improves different abiotic stress tolerance in tobacco, wheat, and rice (Ben- Saad et al., 2015). They also demonstrated that AlSAP transcripts are induced by multiple abiotic stresses, but the ortholog gene of rice OsSAP9 is preferentially induced by cold and heat treatments.
A comparative transcript expression analysis revealed a higher expression of antiporter SOS1 gene in Thellungiella species compared to Arabidopsis (Oh et al., 2010). Similarly, several genes such as SOS2, NHX1, and HKT1 involved in Na + excretion, compartmentation, and diffusion were also expressed at higher levels in Thellungiella compared to Arabidopsis (Taji et al., 2010). To compare the Na + hypersensitivity response, Arabidopsis lines overexpressing either AtHKT1 (A. thaliana) or TsHKT1 (T. salsuginea) were analyzed and delayed root growth was observed in AtHKT1 compared with those expressing TsHKT1 (Ali et al., 2012). The shoot sensitivity was observed in transgenic lines expressing AtHKT1. They also demonstrated a strong salt-dependent up-regulation of TsHKT1 but a strong repression of AtHKT1 expression under salt stress (Ali et al., 2012).
Based on different reports, it may be concluded that halophytic genes are one of the promising candidates to be explored further for producing transgenic plants with a higher level of salt tolerance as compared to glycophytic counterpart genes. Further, halophytes also serve as valuable resources to discover novel abiotic stress responsive genes for improving stress tolerance of crop plants for sustainable agriculture in the saline affected areas.

PERSPECTIVE AND CONCLUSION
Halophytes are more tolerant to abiotic stress because of high differential regulation of the same basic set of stress-responsive genes present among all plants. Furthermore, halophytes exhibited higher expression of a large number of stress-inducible genes under the non-stress condition, suggesting constitutive expression of genes in halophytes. Since different halophytes use different mechanisms to respond the salt stress, a single species cannot be considered as a model species. However, the emergence of a halophyte species as a model plant for the molecular elucidation of corresponding abiotic stress tolerance will enlighten our understanding of the salinity tolerance mechanisms. Identification and isolation of novel salt responsive genes and promoters from different halophytes can be explored for the genetic engineering of crop plants for abiotic stress tolerance using transgenic approach.

AUTHOR CONTRIBUTIONS
AM: Conceived the idea, collected literature and wrote the paper. BT: Helped in revisions. All authors approved this mini-review for the publication. Junior and Senior Research Fellowship to BT is also duly acknowledged. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.