Keywords

1 Introduction

Halophytes are salt-tolerant plants and could thrive under salinity environment, primarily in arid, semi-arid lands, and saline wetlands along the tropical and sub-tropical coasts (Saddhe and Kumar 2019). According to recent estimates, halophytic plants account for about 1–2% of the world’s total flora (Nikalje et al. 2017). They are grouped based on their tolerance and salts demand, as obligate (salt is necessary for their growth and development) and facultative halophytes (can grow in non-saline habitats where the soil is devoid of salt). These plants are remarkably adapted to survive in saline environments and possess salt-responsive genes and proteins to cope with salinity throughout their life cycle (Nikalje et al. 2017). Salinity is a significant problem for crop production, especially in the arid and semi-arid regions as drought and salt stress, together with high temperature, adversely affect plant growth and survival. Therefore, understanding the regulatory mechanism involved in stress responses and identifying stress-resistant genes are necessary to improve the tolerance of crop plants using genetic engineering (Weber 2009). Mishra and Tanna (2017) reported that some halophytic plants like Aeluropus spp., Atriplex spp., Cakile spp., Mesembryanthemum spp., Salicornia spp., Suaeda spp., and Thellungiella spp. are a potential source for the salt-responsive genes and promoters. Studies using the halophyte Thellungiella salsuginea, a model plant with a close evolutionary relationship with Arabidopsis thaliana, revealed the presence of specialized mechanisms, supporting close inspection of genes that might be implicated in salt tolerance (Ali et al. 2013; Wu et al. 2012). Cheeseman (2015) discussed the characteristics of halophytic and glycophytic crop evolution and suggested that in order to facilitate the development of salt-tolerant crops, it is necessary to understand, combine, and manipulate all modes of halophytic evolution at the genome level. Furthermore, the author emphasized that the crops grown on coastal or degraded soils should be considered on priority for salinity-based plant research.

During the past two decades, considerable attention has been given to the identification of cell-based mechanisms of ion and osmotic homeostasis as essential determinants of abiotic stress tolerance in plants (Shabala et al. 2015; Flowers and Colmer 2015; Volkov 2015). From earlier studies, it could be inferred that halophytes tend to use various pathways and physiological mechanisms to sustain under saline environment, and among these cellular responses, Ca2+ and SOS signaling play a significant role. This chapter provides a brief overview of recent progress in the cellular response of halophytes under salinity stress. Furthermore, we discuss different mechanisms coordinately linked with uptake and sensing of Na+, membrane transporter, cellular signaling pathways (Ca2+ and SOS signaling), reactive oxygen species generation, and neutralization, ion homeostasis, synthesis of osmolytes and salt-responsive genes and transcription factors. Lastly, we have summarized some areas that could be addressed in halophytic research.

2 Entry and Sensing of Sodium (Na+) in Halophytes

Halophytes are the salt-loving plants that evolved with unique features such as salinity, water, and nutrient scarcity (Saddhe and Kumar 2019). Our current understanding of salt tolerance mechanisms comes through the glycophytic model plants such as Arabidopsis thaliana and Oryza sativa (Chang et al. 2016; Sivasubramanian et al. 2015). However, with the recent development of tools and techniques in plant biology, researchers can establish a new halophytic model system for understanding salinity stress and their related mechanisms. Some of these halophytic plants include Eutrema salsugineum (Thellungiella halophila), Aster tripolium (Mesembryanthemum crystallinum), and Cakile maritime (Nikalje et al. 2018). A comparative study between glycophytes and halophytes revealed the genetic closeness and quantitative differences in the halophyte (Monihan et al. 2019; Muchate et al. 2016; Volkov 2015). The quantitative differences were lead to the higher expression of essential regulatory salt tolerance genes or proteins in the glycophytes under salt stress (Himabindu et al. 2016; Muchate et al. 2016). There are a substantial number of the reviews and the book chapters available on the halophytic anatomical features, physiology, and biochemical analysis in the salt stress (Himabindu et al. 2016; Nikalje et al. 2017). In this chapter, we have discussed the Na+ sensing mechanism, entry of Na+ into the plant cell, and activation of the cellular signaling cascade. Also, we have shed light on how these mechanisms help halophytes to sustain salinity stress. In the present chapter, salinity means the presence of only Na+ and Cl− ions. The plant root senses the higher Na+ and Cl−concentration in the rhizosphere, which leads to osmotic and ionic stress at the cellular level, disturbed water potential, imbalanced nutrients and minerals, impaired detoxification, antioxidant enzymes, and photosynthetic activity (Shabala et al. 2015).

Plant senses the subtle variation in the environment and responds immediately. They have a well-characterized plasma membrane as well as intracellular localized receptor families that sense and respond to the environmental stimuli. However, there are no scientific records available on Na+ binding receptors and sensing mechanism in plants. It is still a mystery, how plants sense the Na+ level in the rhizosphere and modulate its cellular activity (Shabala et al. 2015). Besides the plasma membrane (PM) receptor theory, another hypothesis was postulated that at first, Na+ entered into the plant cell through various membrane transporters and were sensed by the cytoplasmic Na+ sensor (Shabala et al. 2015). Again, the next question would arise if Na+ is toxic to the plant cell, how they permit Na+ entry inside the cell. There are two possibilities about Na+ uptake in halophytes: (1) they may uptake selectively to build osmoticum, or (2) it may enter non-specifically into the cell through the membrane transporter (Flowers and Colmer 2015). There are several classes of membrane transporters characterized in plants which are involved in specific and non-specific Na+ transporters. It consists of non-selective cation channels (NSCC), including cyclic nucleotide-gated channels (CNGC), and glutamate-like receptors (GLRs), some members of high-affinity K+ transporters (HKTs), the Arabidopsis K+ transporter (AKT1), and high-affinity K+ uptake transporter (HAK) (Shabala et al. 2015).

3 Na+ Membrane Transporter

3.1 Non-selective Cation Channels in Halophytes

In halophytes, roots are facilitated by Na+, through non-selective cation channels (NSCC) including the CNGC, and GLR (Himabindu et al. 2016). The PM possesses different types of non-selective cation channels (NSCC), which are permeable to a broad range of monovalent as well as divalent cations such as Na+, K+, and Ca2+ (Demidchik and Maathuis 2007). They respond to a wide range of external stimuli such as reactive oxygen species (ROS), pathogen-specific antigen, cyclic nucleotides (cGMP and cAMP), and amino acids. They are also involved in physiological functions such as stress response, plant growth and development, nutrient uptake, and Ca2+ signaling (Demidchik and Maathuis 2007). Moreover, the low level of ROS is generated by depolarization of the PM and NADPH oxidase activity at the apoplast region and if further involved in the regulation of PM transporter activity (Demidchik and Maathuis 2007). Comparative studies on salt tolerance mechanism between Arabidopsis thaliana and Thellungiella halophila revealed the variation in Na+ and K+ levels (Volkov and Amtmann 2006; Wang et al. 2006), where T. halophila maintained a fourfold higher PK/PNa ratio using the root localized depolarization activated NSCC-like channel (Volkov and Amtmann 2006). Moreover, the lower PK/PNa was recorded for T. halophila for other classes of Na+ influx NSCCs channels (Volkov and Amtmann 2006).

The cyclic nucleotide-gated channel (CNGC) seems to get activated by cAMP or cGMP ligands under stress conditions (Duszyn et al. 2019). It has two C-terminal domains, calmodulin-binding domains (CaMB) and cyclic nucleotide-binding (CNB) domain, and both faced toward the cytoplasm. Whereas, the single N-terminal transmembrane (TM) domain faces the apoplast region (Duszyn et al. 2019). The gene expression analysis of the CNGC in A. thaliana and Amaranthus hypochondriacus showed significant up- and downregulation during salinity stress (Duszyn et al. 2019). On the other hand, in halophytic plants, there are no such systematic studies on CNGC gene family and their roles in salinity stress. It could be a potential investigating area to explore the roles of halophyte NSCC members in salinity stress.

3.2 High-Affinity K+ Transporter (HKT)

The plant HKT family is the cation-specific transporter, subdivided into two subcategories based on the selectivity of ion and pore-forming motifs, including class I and class II. The class II members have a “GGGG” motif, which is reported in monocots. Similarly, the class I members have an “SGGG” motif, which is present in both plant groups (monocot and dicot) (Ali and Yun 2017). The class I subfamily is responsible for Na+ uptake, but class II is responsible for Na+ and K+ uptake (Ali and Yun 2017). However, in the class I HKT motif, the G residue was replaced by S residue, which restricts K+ permeability due to steric hindrance (Ali and Yun 2017). The subcellular localization of the class I HKT transporters was reported in the xylem boundary of roots and shoot tissues. Further, they mediated the Na+ transport back to the xylem from xylem parenchymatic cells. Arabidopsis contains only one isoform of class I HKT; in contrast, monocots have multiple HKT isoform. The Arabidopsis HKT1 is involved in long-distance Na+ transport through xylem and phloem tissues (Sunarpi et al. 2005). In rice, HKT2;1 transporter selectively facilitates the Na+ uptake during low levels of K+ and Na+ in the soil (Horie et al. 2009). The HKT transcript abundance was seen in a halophytic grass Aeluropus lagopoides at a different time point under salt stress (Sanadhya et al. 2015). AlHKT2;1 transcript level was found to be upregulated in the shoot at 12 and 24 h, while significant transcript abundance was recorded during K+ starvation in the roots (Sanadhya et al. 2015). Zhao et al. (2017) reported the four HKT gene members in a halophytic grass Kochia sieversiana, where no significant transcript abundance was observed for HKT genes under the salt stress (Zhao et al. 2017).

4 Salt Overly Sensitive Signaling Pathways

The plant cell responds to excess cytoplasmic Na+ by activating the Ca2+ signaling cascade, which helps to maintain Na+ level inside the cell (Saddhe and Kumar 2019). The SOS system is involved in the efflux of excess Na+, loading of Na+ in the xylem, and sequestration into the vacuole using sodium/hydrogen exchanger (NHX). The key components of SOS pathways are SOS1 (plasma membrane Na+/H+ antiporter), SOS2 (a member of protein kinase), and SOS3 (SCaBP8, calcium sensor) (Nikalje et al. 2017). Moreover, two members, SOS4 and SOS5, are included in the SOS pathways reporters. The SOS4 (pyridoxal kinase) seems to be involved in the biosynthesis of pyridoxal-5-phosphate (vitamin B6) and played an important role in Na+ and K+ homeostasis in plants, while SOS5 is a putative cell surface adhesion protein required for normal cell expansion. Moreover, they are involved in the maintenance of cell wall integrity and architecture during salt stress (Fig. 1).

Fig. 1
figure 1

Na+ sensing and uptake mechanism in halophytes. (1) Membrane transporter: there are several classes of Na+ membrane transporter, including non-selective cation channels (NSCC), cyclic nucleotide-gated channels (CNGC), glutamate-like receptor (GLR), and high-affinity K+ transporter (HKT). CNGC channels were activated by cyclic nucleotide monophosphate (cAMP and cGMP) and involved in the transport of Ca2+ and Na+. Similarly, activation of NSCC transporter was trigger by ROS species (H2O2) and membrane depolarization. Ca2+ ions uptake depends on the Na+ level inside the cell, which will trigger the plasma membrane, as well as endo-membrane localize, Ca2+ transporters. The PM localized Ca2+ ATPase and TPC Ca2+ channels were involved in Ca2+ uptake from the apoplast region. Similarly, the vacuolar Ca2+/H+ channel released the stored Ca2+ ions into the cytoplasm region. (2) Activation of Ca2+ dependent and SOS pathways: Ca2+ acted as a secondary messenger and interacted with protein consisting of Ca2+ binding motif, such as SOS3/CBL and calmodulin (CaM). Activated SOS3/CBL protein interacted with SOS2/CIPK (protein kinase) and formed a complex, which activated the multiple downstream targets such as SOS1 (PM Na+/H+ exchanger), vacuolar Na+/H+ exchanger, vacuolar Ca2+/H+ channels, and H+-ATPase. Activated SOS1 and vacuolar NHX maintained the cellular Na+ level by pumping surplus Na+ either outside the cell or inside the vacuole. Moreover, the SOS2 phosphorylated the CBL10 protein and this complex will activate the nucleoside diphosphate kinase 2 proteins (NDPK 2). Further, the NDPK 2 activated the ROS signaling cascade. (3) MAPK pathways: higher Ca2+ level triggered the phospholipase D enzyme, which will produce phosphatidic acid (PA). The PA was activating MAPK6 kinase, which will act on the SOS1 exchanger

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However, SOS3 protein has a small EF-hand motif, myristoylation, and shares sequence similarity with the yeast and animal calcineurin B protein. It requires Ca2+ and myristoylation for function in salt tolerance. The SOS3/CBL4 (calcineurin B-like 4) senses the Ca2+ spike in response to excess cytoplasmic Na+ level. The activated SOS3 protein binds and interacts with SOS2 (CIPK24/SOS2). The activated complex of SOS3 and SOS2 gets phosphorylated along with SOS1; this triggers the SOS1 activity initiating an efflux of excess Na+ from the cell (Nikalje et al. 2017). Thus, the CIPK24/SOS2 and CBL4/SOS3 complex regulates the PM-bound Na+/H+ antiporter (SOS1) activity in roots and confreres the salt tolerance (Kudla et al. 2010), whereas CBL10-CIPK24 complex regulates the shoot SOS1 activity in salt stress (Huang et al. 2019). Interestingly, the CIPK24 can phosphorylate the CBL10 protein and stabilize the complex at PM, thereby conferring salt tolerance (Huang et al. 2019). Moreover, CIPK24/SOS2 interacts with nucleoside triphosphate kinase 2 (NDPK2), further maintaining the ROS signaling cascade and catalyzing the enzyme activity (CAT2 and CAT3) (Verslues et al. 2007). These findings suggested that CIPK24/SOS2 kinase is an important regulatory node in the stress signaling network. Besides the SOS signaling components, CIPK orthologs were identified, which worked independently from the SOS pathway required for salt tolerance (Huang et al. 2019). The CIPK controls the K+ transporter, including AKT1 (Huang et al. 2019). However, the regulation of K+ homeostasis through the SOS pathway was poorly understood.

The breakthrough work on a model halophyte plant Thellungiella halophila demonstrated the importance of SOS1 in Na+ homeostasis (Oh et al. 2009). Further, the knockdown studies confirmed their role in halophytic characteristics of T. halophile (Oh et al. 2009). Halophytes seem to have much efficient mechanisms as compared to the salt-sensitive species in order to remove the excess Na+ from the cytosol through plasma membrane NHX (SOS1) (Bose et al. 2014). They have achieved salt tolerance potential through cumulative action of several mechanisms, including Ca2+ and SOS signaling components, low-level ROS production, and efficient antioxidant systems (Bose et al. 2014). The Mesembryanthemum crystallinum is a facultative halophyte that exhibits no detectable SOS1 expression in roots, but gene expression has been observed in leaves under salt stress (Cosentino et al. 2010). This result suggested that the roots localized SOS1 has no major role in xylem loading of Na+ but involved in the maintenance of low cytosolic Na+ in the leaves under salt stress (Cosentino et al. 2010). Katschnig et al. (2015) compared the effects of salinity on an ion accumulation and expression of salt tolerance genes in the salt accumulating halophyte Salicornia dolichostachya and the taxonomically related glycophytic species, Spinacia oleracea. The S. dolichostachya species showed constitutively high SOS1 expression, but no detectable expression of HKT1;1 gene was observed (Katschnig et al. 2015). These findings suggest that the S. dolichostachya species accumulate the highest concentration of salt in its shoots through SOS1-mediated Na+ loading in the xylem, which transfers excess Na+ toward shoot and suppressed the HKT1;1 activity in salt stress (Katschnig et al. 2015).

Eukaryotic 14-3-3 proteins are dimeric, highly conserved, and general regulatory proteins (Camoni et al. 2018). These proteins interact with diverse proteins, including membrane receptors, phosphatases, and kinases; they regulate a broad range of physiological processes such as regulation of cell cycle, mitogen signaling, and programmed cell death (Camoni et al. 2018). In Arabidopsis thaliana, 14-3-3 proteins interact with SOS2 kinase, a member of the SOS pathway, further inhibiting its activity (Zhou et al. 2014). In salt stress, Arabidopsis thaliana 14-3-3 proteins are involved in the regulation of SOS pathways by inhibiting SOS2 kinase activity; when dissociated from SOS2 protein kinase, they triggered the SOS pathways (Tan et al. 2016). Moreover, in the future, it will be interesting to explore the physiological function and roles of 14-3-3 proteins under salinity stress in halophytes.

According to Yu et al. (2010), the Arabidopsis SOS1 activity in response to salt stress seems to be regulated by phospholipase D (PLD) and phosphatidic acid (PA) through the regulation of mitogen-activated protein kinase (MAPK). These findings have revealed new directions for research on halophytes and also will be interesting to check the correlation between phospholipase D (PLD) and SOS1 regulation. In addition to SOS1 regulation, activated SOS2 kinases seems to regulate the Ca2+/H+ transporter, vacuolar Na+/H+, and vacuolar H+-ATPase activity (Batelli et al. 2007). The SOS2 kinase also interacts with a protein phosphatase 2C (PP2C), the catalases (CAT2 and CAT3), nucleoside diphosphate kinase 2 and the flowering time regulator GIGANTEA (GI) (Kim et al. 2013). As compared to glycophytes, very preliminary scientific information is available on the halophytic SOS signaling mechanism and its diverse functions. It will be interesting to investigate thoroughly halophytic SOS pathways in salt stress and under normal physiology.

As we mentioned earlier, SOS2 kinase interacts with vacuolar NHX transporter, which plays a crucial role in the sequestration of excess Na+ into vacuole using proton gradient energy. In the genome of Arabidopsis thaliana, there are eight NHX genes which could be classified based on the localization, including the plasma membrane (NHX7/SOS1 and NHX8), endosome (NHX5 and NHX6), and vacuole (NHX1, NHX2, NHX3, and NHX4) (Himabindu et al. 2016). The first plant vacuolar Na+/H+ antiporter gene was isolated from the Arabidopsis thaliana, which showed significant transcript abundance in salt and abscisic acid (Apse et al. 1999). Further, halophytic NHX gene isolation and characterization was reported in various species, including Mesembryanthemum crystallinum, Atriplex gmelinii, Populus euphratica, Salicornia brachiata, Suaeda salsa, Halostachys caspica, Zygophyllum xanthoxylum, and Rhizophora apiculata (Mishra and Tanna 2017; Chauhan et al. 2000; Saddhe and Kumar 2019; Ye et al. 2009). In Mesembryanthemum crystallinum, NHX gene expression analysis showed significant up-regulation in leaves and stems (Chauhan et al. 2000). This finding showed that McNHX was involved in the sequestration of excess Na+ into vacuoles of leaf tissues under the stress conditions (Chauhan et al. 2000). There were six NHX family members reported in the Populus euphratica, of which PeNHX1, 3, and 6 transcripts levels were found significantly higher in the roots, stems, and leaves, as compared to the PeNHX2, 4, and 5 (Ye et al. 2009). Further, all PeNHX members were functionally characterized in yeast mutants using complementation assay, which underscored their functional roles in salt stress (Ye et al. 2009).

5 Production and Maintenance of Reactive Oxygen and Nitrogen Species Under Salt Stress

The salinity stress seems to affect the redox homeostasis in plants and generated the excess reactive oxygen species (ROS) (Demidchik 2015; Nikalje et al. 2018), where excess ROS causes oxidative stress, which is deleterious to cell components (Demidchik 2015). In contrast, ROS also acts as a sensing and signaling molecule in several stress conditions (Saddhe et al. 2019). The ROS consisted of free radical and non-radical molecules, including superoxide radicals (O2−), hydrogen peroxide (H2O2), and hydroxyl radicals (∙OH) (Table 1). The main site of ROS production is in the cytoplasm and subcellular compartments, including chloroplasts, mitochondria, and peroxisomes (Bose et al. 2014). Moreover, ROS are produced in the apoplastic region by NADPH oxidases and cell wall-associated peroxidases (POXs) (Bose et al. 2014). ROS is responsible for lipid peroxidation, DNA damage, loss of protein activity due to denaturation, carbohydrate oxidation, pigment breakdown, and reduced enzymatic activity (Bose et al. 2014). Substantial work has been done on halophytes to determine the antioxidant defense mechanism and its importance in maintaining proper plant growth under various abiotic stress conditions.

Table 1 List of ROS and RNS free radicals and non-radicals
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Moreover, plants have evolved with an antioxidant enzyme system for scavenging ROS molecules and reducing their detrimental effects. Halophytes have enzymatic and non-enzymatic pathways for ROS elimination. There are two possibilities on halophytes antioxidant enzyme system either (1) they are the most efficient antioxidant system to nullify ROS or (2) halophytes maintained low ROS production. The enzymatic pathways consist of sodium dismutase (SOD), peroxidase (POX), ascorbate peroxidase (APX), and catalase (CAT), while redox regulatory enzymes include monodehydroascorbate reductase (MDAR), glutathione reductase (GR), glutathione peroxidase (GPX), glutathione S-transferases (GST), thiol peroxidase type II peroxiredoxin (Prx), and APX (Bose et al. 2014). However, various ROS species such as 1O2 and OH· do not scavenge effectively by enzymatic activity. Thus, plants use alternative non-enzymatic strategies to eliminate highly toxic ROS molecules. The major non-enzymatic action consists of ascorbate, glutathione, carotenoids, and tocopherol antioxidants (Nikalje et al. 2017). There are many reports available on the characterization of halophyte antioxidant enzymes and their roles in ROS scavenging (Muchate et al. 2016; Zhao et al. 2017). In Sesuvium portulacastrum, physio-biochemical analysis of a facultative halophyte was maintained with the help of antioxidant enzyme activity (CAT, GR, SOD, APX, and GPX) under the salt stress (Muchate et al. 2016). However, most of the transcript and RNA-Seq analysis reported the upregulation of antioxidant enzymes in salt stress (Zhao et al. 2017).

Similarly, reactive nitrogen species (RNS) may also enhance under stress, a condition called “nitrosative stress” that affects cellular functions (Saddhe et al. 2019). Nitric oxide (·NO) was a source of RNS species that served as a key signaling component in stress tolerance mechanisms (Frungillo et al. 2014). RNS species consists of radicals such as a nitric oxide (·NO), nitrogen dioxide (·NO2), and non-radical members were S-nitrosothiols, peroxynitrite (ONOO−), nitroxyl anion (NO−), nitrate (NO3−), nitrosonium cation (NO+), dinitrogen trioxide (N2O3), dinitrogen tetroxide (N2O4), nitryl chloride (NO2Cl), and nitrous acid (HNO2) (Saddhe et al. 2019) (Table 1).

6 Ca2+ Signaling in Salinity Stress

Calcium (Ca2+) is a well-known plant micronutrient and a secondary messenger in several signaling pathways, including abiotic stress (Demidchik et al. 2018; Seifikalhor et al. 2019). A quick and transient spike of cytosolic Ca2+ level in response to external stimuli is a conserved signaling cascade across the plant kingdom (Demidchik et al. 2018). The stimuli generate the cellular spatiotemporal pattern of the Ca2+ signature, which is proportional to stimuli that determine their specificity through activating downstream targets. For the regulation of cytosolic Ca2+ level, the Ca2+ transporters are involved including, CNGCs, doubled-pore Ca2+ channels (TPCs), Ca2+-ATPase, and a vacuolar H+/Ca2+ antiporter (CAXs) (Demidchik et al. 2018; Seifikalhor et al. 2019). The Ca2+ burst transduces the external signal to the downstream targets, including calmodulin (CaM), CaM-like proteins (CMLs), calcineurin B-like proteins (CBLs), CBL interacting protein kinases (CIPKs), and Ca2+-dependent kinases (CDPKs). The CaM, CML, CBL, and CDPK proteins mostly consist of the EF-hand Ca2+-binding motif. The CIPK is a novel plant-specific serine-threonine protein kinase gene family, which interacts with CBLs to form the CBL-CIPK complex. The activated CBL-CIPK complex regulates the expression of several downstream stress targets such as SOS pathways, and salt responsive genes and transcription factors. Recently, several CBL-CIPK signaling components were identified and were functionally characterized in glycophytic and halophytic plants (Kudla et al. 2010).

Moreover, gene duplication events can help in expanding gene families through segmental duplication, tandem duplication, and retro-transposition (Kondrashov 2012). A new member of the gene family could acquire either sub-function or novel function of the existing gene (Kondrashov 2012). Recently, a gene duplication event was observed in the Eutrema salsugineum species CBL10 gene, which was a salt-tolerant relative of A. thaliana (Monihan et al. 2019). The authors confirmed that the gene duplication event in the E. salsugineum was associated with an adaptation to salt stress (Monihan et al. 2019). Further, the downregulation of EsCBL10a and EsCBL10b genes showed a decreased plant growth rate, and even a significantly lowered growth rate when both genes were downregulated. The overall research suggested that the duplication of the CBL10 gene augmented calcium signaling in salt stress, which helps them to survive in extreme salinity (Monihan et al. 2019). According to Wang et al. (2013) salt tolerance characteristics in Populus euphratica tree species were evolved with gene family expansion, through duplication or a higher expression of salt stress-related genes, including ion transport and homeostasis. Tandem duplication of the HKT gene and upregulation of CIPK1 were to a greater extent reported in P. euphratica than in Populus tomentosa (Wang et al. 2013). Functional characterization of the HbCIPK2 gene from halophyte plant Hordeum brevisubulatum revealed its importance in salinity stress when complemented with the Arabidopsis sos2-1 mutant phenotypes (Li et al. 2012). This finding underscored the genetic closeness and similarity between the SOS pathway in glycophytes and halophytes. Moreover, the Ca2+ signaling is a significant adaptive characteristic in halophytes over glycophytes.

7 Role of Mitogen-Activated Protein Kinase Pathway in Salt Stress

In eukaryotes, mitogen-activated protein kinase signaling cascades are highly conserved and are responsible for regulating physiological functions, including plant growth and development, hormonal response, biotic, and abiotic stress signaling (Agarwal et al. 2010). There was a substantial scientific research gap between the halophyte and the glycophytes MAP kinase signaling pathways in salinity stress. Earlier glycophytic studies on MAP kinase signaling underscored their importance in salinity stress (Agarwal et al. 2010). A full-length mitogen-activated protein kinase kinases gene was isolated from a halophyte plant species Salicornia brachiata, which belongs to MAPKK D-group, and shared similarity with Nicotiana benthamiana (NbMKK1) and Lycopersicon esculentum of (LeMKK4) genes (Agarwal et al. 2010). Further, the authors performed the transcript abundance in dehydration, cold, and salt tolerance, but significant expression was observed in the cold stress (Agarwal et al. 2010). Recently, Wang et al. (2017) isolated a MAPKK gene from a salt-tolerant plant species Chenopodium album belonging to the MAPKK group A. The transcript abundance of CaMKK1 showed a significant upregulation pattern under the salt and drought stress (Wang et al. 2017). Ectopic expression of CaMKK1 in Escherichia coli and tobacco showed that with the increase in the salt and drought tolerance in a bacterium, and transgenic tobacco, significant germination rate and seedling growth was observed (Wang et al. 2017). Similarly, Rehman et al. (2020) targeted a halophytic plant Salicornia europaea and performed cloning and functional characterization of the SeMAPKK gene. An evolutionary relationship of SeMAPKK showed that it belongs to the MAPK group D, while its transcript abundance reported significant expression pattern at 0.75 M of salt stress and downregulation at 1.0 M of salt concentration (Rehman et al. 2020). Further, an overexpression study of the SeMAPKK gene in Arabidopsis thaliana showed significant salt tolerance and growth as compared to the WT plants (Rehman et al. 2020). Moreover, the MAPK act as a convergent point for signaling pathways; they responded to physiological functions, biotic, and abiotic stress. It will be interesting to explore halophytic MAPK kinase and their roles under salinity stress.

8 Salt-Responsive Transcription Factor

Transcription factors are key elements that provide a carrier for genetic engineering, as they play an important role in the regulation and modification of different stress-responsive genes such as salinity. In this point, we will review major plant transcription factors (TF), like the NAC (NAM, ATAF1/2, and CUC2), dehydration-responsive element-binding protein (DREB), basic-domain leucine zipper (bZIP), ethylene-responsive factor (ERF), and WRKY (conserved sequence WRKYGQK and a zinc finger motif CX4–7CX22–23HXH/C) exhibit higher correlations with salinity (Saddhe et al. 2017b). Moreover, TFs strictly control the expression of the respective gene, as the post-transcriptional regulation of TFs depends on the modulation in the expression of the target genes. In this process, an important role in signal transduction, which is played by TFs, they interact with cis-regulatory sequences further modulating the expression of a series of concerned genes against different abiotic stresses. In recent years, many studies have reported the involvement of NAC TFs in biotic and abiotic (Nuruzzaman et al. 2013) stress responses in plants. The NAC TFs confer abiotic stress tolerance to transgenic plants, for instance, transgenic rice plants overexpressing OsNAC3, OsNAC6, or OsNAC14 enhanced drought tolerance (Fang et al. 2015; Lee et al. 2017; Shim et al. 2018), while overexpression of OsNAC6 induced tolerance to high salt, drought, and blast disease in rice (Nakashima et al. 2007). Studies on halophytes revealed the overexpression of TsNAC1 in Thellungiella halophile as well as in Arabidopsis that could enhance abiotic stress resistance, particularly salt stress tolerance (Liu et al. 2018). Moreover, the RNA-sequencing (RNA-Seq) comparison between wild-type ThNAC7 of Tamarix hispida and ThNAC7-transformed Arabidopsis showed that more than 40 known salt tolerance genes that might be regulated by ThNAC7, including stress tolerance-related genes and TF genes. The SlNAC8 gene from Suaeda liaotungensis demonstrated enhanced tolerance to drought and salt stress in Arabidopsis plants, further suggesting that in transgenic plants, it could enhance the expression of stress-responsive genes (Wu et al. 2018).

Another important transcription factor is DREB. It plays an important role in regulating the expression of stress-inducible genes under abiotic stresses. The expression of SsDREBa and SsDREBb genes in S. salsa roots and leaves was remarkably induced by high-salt and dehydration treatments, further indicating them as novel stress-responsive transcription factors, through ABA-independent pathways (Sun et al. 2014). Also, the overexpression of DREB transcription factor may improve stress tolerance in plants, as of SsDREB cDNA in transgenic tobacco plants exhibited an improved salt and drought stress tolerance in comparison to the non-transformed controls (Zhang et al. 2015). In Tamarix hispida, ThDREB exhibited improved plant salt and drought tolerance in comparison with the empty vector-transformed lines (Yang et al. 2017).

Among the important TFs, basic leucine zipper proteins (bZIPs) are one of the large family, where bZIP has been identified as one of the TF having great potential in improving crop tolerance against salinity stress. Like other transcription factors, the bZIPs play important roles in plant growth and developmental processes; apart from this it also responds to numerous other abiotic stresses including drought, salinity (Liu et al. 2014), and cold stresses (Jakoby et al. 2002). Increased tolerance to drought and salinity was observed in Arabidopsis due to over-expression of a ThbZIP from the woody halophyte Tamarix hispida (Ji et al. 2013). Moreover, Wang et al. (2010) reported another bZIP gene (ThbZIP1) from Tamarix hispida that showed higher salt resistance in transgenic tobacco by avoiding the accumulation of cellular ROS, cell death, and minimizing water deficiency. In the halophytic relative of Arabidopsis “Lobularia maritima,” the salinity stress resulted in the activation of gene expression involved in cytoplasmic ion homeostasis and osmotic adjustment, viz., AtHKT1, AtSOS1, AtPIP2.1, and glutamine synthetase (Yang et al. 2009). Overall, bZIP tends to play a central role in conferring stress tolerance as reviewed by Golldack et al. (2011), further stating that several transcription factor families (bZIP, MYB, WRKY, AP2/ERF, NAC, and zinc finger proteins) are involved in the plant adaptation to the saline conditions. Golldack et al. (2011) presented a model that bZIP receives a signal of stress from MYB and regulates zinc finger proteins, MYB AP2/ERF, and NAC, located downstream of the signaling pathway.

Another group of plant-specific TFs is ethylene-responsive factors (ERFs) that involved in multiple biological processes, especially in abiotic stress tolerance. However, the ERFs from halophytes that are involved in salt stress have not received much attention. Qin et al. (2017), investigated and characterized subfamily members of ERF TFs from Tamarix hispida, ThCRF1 with the response to salt stress. Overexpression of ThCRF1 in T. hispida significantly improved tolerance to salt-shock-induced stress; by contrast, RNAi-silence of ThCRF1 significantly decreased tolerance to salt-shock-induced stress (Qin et al. 2017).

WRKY transcription factors are one of the largest families of transcriptional regulators in plants with their known functions associated with growth, seed development, leaf senescence, and responses to abiotic and biotic stresses (Rushton et al. 2010). Studies showed that they aid in responses to various abiotic stresses like drought, salinity, osmotic stress, heat, and cold (Banerjee and Roychoudhury 2015). The WRKY genes are quite common in plants, the Arabidopsis contains 74 WRKY genes, while in rice, more than 100 have been recorded. In Arabidopsis, the function of WRKY33 TF is targeting the salt responsive downstream genes that helps in detoxification of ROS and regulation of LOX1 (lipoxygenase), GSTU11 (glutathione-S-transferase), and peroxidases; along with this WRKY25 and WRKY33 are two WRKY factors which have shown to enhance salinity stress tolerance (Jiang and Deyholos 2009). In rice, Cai et al. (2014) found that group II transcription factor of WRKY, i.e., WRKY58 increased the level of salt tolerance. In halophytes, the herbaceous perennial herb Iris lactea var. chinensis was studied for its IlWRKY1 gene, where transcriptional levels revealed increased expression of IlWRKY1 under NaCl stress in shoots, further achieving the highest level suggesting that IlWRKY1 may be involved in sodium salt responses (Tang et al. 2018).

9 Salt-Responsive Genes

As in salt environments, plants have to deal with osmotic stress that reflects water deficiency, followed by ion-specific stress resulted from altered ion concentrations. The responses of halophytes to high salinity can be divided into two strategies, salt tolerance and salt exclusion. The salt tolerance mechanism includes a reduction of the Na+ influx, compartmentalization, and excretion of sodium ions (Flowers and Colmer 2015), while salt exclusion mechanism consists of secretion, shedding, and succulence (Mishra and Tanna 2017). Hereby, we have tried to review the studies done on salt tolerance mechanisms and discussed various salt responsive genes of halophytes in an integrative way. During this decade, efforts have been made to isolate salt-responsive genes from several halophytes, followed by their functional validation through transgenic approaches. The overexpression of several halophytic genes, under the control conditions, could enhance abiotic stress tolerance in the glycophytic recipients (Table 2). In recent years, the Salicornia brachiata seems to be the most studied species; its transgenic recipients were assessed for their salt tolerance potential. Ricinus communis (Patel et al. 2015) and Cuminum cyminum (Pandey et al. 2016) ectopic expression of the sodium/hydrogen exchanger gene SbNHX1 demonstrated enhanced salt tolerance of transgenic plants as compared to wild type (WT) by modulating physiological process. Similarly, in transgenic tobacco, the SbNHX1 promoter tends to possess two leaf-specific enhancer motifs and important stress-responsive cis-regulatory motifs like GT1, MBS, LTR, and ARE, inducing transcript expression either in the control or salinity stress conditions (Tiwari et al. 2019). Moreover, significantly enhanced salt tolerance was observed in transgenic tobacco plants from the KvNHX1 genes of Kosteletzkya virginica halophyte. This transgenic variety exhibited better growth, more chlorophyll, higher antioxidant enzyme activities, and osmoregulation capability (Wang et al. 2018). Overall, transgenic plants that have received the NHX1 gene from a respective halophyte could thrive well under salinity stress as compared to WT plants, as evidenced by higher photosynthetic pigments and lower physio-biochemical indicators (electrolyte leakage, proline, and malondialdehyde) and enhanced antioxidant enzyme activities and osmoregulation capability. In case of Salt Overly Sensitive (SOS1), which are considered as plasma membrane antiporters, it was observed that in Arabidopsis SOS1 mutant plants expressing SpSOS1 from Sesuvium portulacastrum grew better and had a lower Na+/K+ ratio than that of the sos1 mutant and WT plants (Zhou et al. 2018). Similar observations were recorded in transgenic recipient Gossypium hirsutum (cotton) GhSOS1-VIGS (virus-induced gene silencing) expressing TrSOS1 from Tamarix ramosissima. Moreover, cotton seedling exhibiting GhSOS1-VIGS maintained enhanced root vigor and leaf relative water content (Che et al. 2019).

Table 2 Salt-responsive halophytes genes
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The transporter family, such as high-affinity K+ Transporter (HKT1) type, has received great attention that played a crucial role in Na+ and K+ homeostasis. It has also been observed that knockout or downregulation of HKT1 leads to sensitivity to NaCl in both glycophytes (Arabidopsis) and halophytes (Thellungiella salsuginea) (Ali et al. 2013; Sunarpi et al. 2005). Ali and Yun (2016) analyzed T. salsuginea TsHKT1;2 and observed that TsHKT1;2, one of the three copies of HKT1, acts as a potassium transporter, even under salt stress. Usually, most of the HKT1 sequences, including Arabidopsis AtHKT1, contain asparagine (N), but TsHKT1;2 has a conserved aspartic acid (D) residue in the 2nd pore-loop domain. The author found that Arabidopsis AtHKT1-1 mutant plants complemented by TsHKT1;2 under native AtHKT1 promoter were more tolerant to salt stress, while substitution of Asp (D207) by Asn (N) significantly reduced resistance to salinity. Moreover, downregulation of EsHKT1;2 in E. salsuginea leads to hypersensitive phenotypes under K+-deficient conditions. Another Arabidopsis halophytic relative is Eutrema parvula, which has two HKT1 genes encoding EpHKT1;1 and EpHKT1;2. In the high salinity, the EpHKT1;2 transcript level increases rapidly; comparatively, the EpHKT1;1 transcript increased more slowly in response to salt treatment (Ali et al. 2018).

Along with the HKT transporter, High-affinity K+ (HAK) is responsible for Na+ and K+ homeostasis and probable salt secretion. Sanadhya et al. (2015) reported that the K+/Na+ ratio in shoots and roots of Aeluropus lagopoides were maintained at the optimum salt concentration (100–300 mM NaCl), probably due to the strong activity of K+ transporters like HKT (Class II) and HAK. Moreover, some halophytic grasses like Phragmites karka (Abideen et al. 2014) and Puccinellia tenuiflora (Yu et al. 2011) exhibited higher cytoplasmic K+/Na+ ratios under high salinity. The higher expression of the HAK transcript during salt stress and K+ starvation can be correlated to its role in salt tolerance (Sanadhya et al. 2015).

10 Transcriptomic Analysis Revealed Salt Stress-Responsive Genes

The gene expression patterns of candidate genes involved in salt tolerance could be identified using the transcriptomics approach. In this section, the current status of transcriptome analysis in halophytes has been reviewed, along with the implications of salt tolerance mechanisms with the identified genes (Table 3). For a better understanding of the salt stress mechanism in plants, halophytes could be the potential source as they are the natural flora of saline habitats. Halophytes have been under-examined until recently, and their utilization may allow the production of useful crops on salty soils. The most extensively studied halophytes belong to the genera Suaeda (Table 3). Suaeda fruticosa in optimal salt concentration (300 mM) exhibits upregulation of zeaxanthin epoxidase, aquaporin TIP2, dehydration responsive protein, and glutathione S-transferase, while the other three targets nitrate reductase, putative protein phosphatase and calcineurin B-like (CBL) showed downregulation (Diray-Arce et al. 2015). The mechanism of salinity tolerance involves control of ionic homeostasis, where the Na+ influx transporter (HKT) and the tonoplast Na+/H+ antiporter (NHX) are responsible for Na+ homeostasis and vacuolar compartmentalization. In case of Suaeda fruticosa and Suaeda glauca, HKT and NHX antiporter did not exhibit significant expression in response to salt stress (Diray-Arce et al. 2015; Jin et al. 2016), while Suaeda maritima and Spartina alterniflora showed significant upregulation by maintaining ion homeostasis (Bedre et al. 2016; Gharat et al. 2016). Also, Na+/K+ ratio is essential for salinity tolerance in plants; thus, salt stress increases Na+, which leads to loss of K+. Spartina alterniflora maintained a higher K+ accumulation under high salt stress via the active gene expression dynamics of SaAKT2, SaKAT2, SaSKOR, and SaNHX2. Moreover, the SaNHX2 showed significant upregulation under high salt stress, further indicating the involvement of SaNHX2 in the K+ reservoir of Spartina (Ye et al. 2019). In S. virginicus, exclusion of ions from roots and secretion of ions from salt glands helped in the achievement of salt balance (Yamamoto et al. 2015). Along with Na+/H+ antiporters and K+ ions that are essential to achieve ionic balance, the aquaporin tonoplast intrinsic proteins were also reported to be associated with the salt glands and were upregulated under salinity in S. fruticosa and S. virginicus (Diray-Arce et al. 2015; Yamamoto et al. 2015).

Table 3 Studies on transcriptome analysis in halophytes for identification of genes/transcripts involved in salt stress
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However, Gharat et al. (2016) suggested that complexity leading to salt tolerance could be due to enhanced expression of transcription factors, such as NAC and HD-Zip, and by the enhanced expression of the various hormone-responsive factors, which might regulate a wide range of chemical reactions and molecular events leading to salt tolerance. Remarkably, Yamamoto et al. (2015) found 19 genes for bZIPs in the transcriptome of S. virginicus that were not found in glycophytes, rice, or Arabidopsis. Interestingly, five of the bZIP genes showed root-specific salt-responsive expression. Feng et al. (2015) targeted two auxin responsive factors (ARF genes) and one NAC transcription factor in Salicornia europaea; all of those responsible miRNAs were downregulated, indicating the release of miRNA-mediated repression of auxin signaling by salt may represent an important mechanism. In case of Na+/H+ antiporter activity, they suggested that UBP16 is required for salt tolerance, where suppression of respective miRNA (seu-miR396h/i) was observed due to long-term salt treatment resulting in increased expression of UBP which speculates that miR393 is a negative regulator of salt tolerance (Feng et al. 2015).

Moreover, to reflect timely signal transduction, abscisic acid (ABA) genes might play an important role in the earlier stage when plant is suffering from salt stress (Jin et al. 2016). Zhang et al. (2014) characterized salt tolerance mechanisms underlying in Karelinia caspica, an Asteraceae halophyte, where they reported that several genes that are differentially expressed could be involved in the signaling pathway of the plant hormone ABA, including ABA metabolism, transport, and sensing as well as the ABA signaling cascade (Zhang et al. 2014). Besides ABA, several DGEs are involved in the signaling of other hormones, and this indicates that those many DGEs play an important role in multiple hormones signaling in K. caspica in responses to the salt stress (Zhang et al. 2014).

11 Conclusion

This review provided an overview of what has been learned so far about cellular signaling and salt stress-responsive genes in halophytes. Differential expression/accumulation of specific stress tolerance genes, proteins, and metabolites have been explored in halophytes under salt stress. Moreover, the effective use of “omics” techniques have helped to reveal unique stress-responsive genes and associated pathways in halophytic research. It revealed the significant upregulation and downregulation pattern of various class membrane transporter, Ca2+ and SOS signaling cascade, antioxidant systems, and different class of transcription factors. The literature review suggested that the Na+ sensing and uptake mechanism is complex and not yet fully understood. It will be a potential research area to explore Na+ sensor, transporter, and related signaling cascade in halophytes. Highly evolved Ca2+ signaling cascade in halophytes is helping to improve the salinity signaling and tolerance. On the other hand, studies focused on the importance of quantitative improvements of genes functions, gene duplication events, and their efficiency in salinity stress. It will be interesting to explore gene duplication events, which favor halophytic characteristics. Besides, it will be important to shed light on how many gene families related to salt stress experience the gene duplication events in arid desert halophytes. As already highlighted by researchers, we also suggest that studies defining the regulatory mechanisms operating differently in glycophytes and halophytes in response to high salinity stress would be interesting in arid desert environments.