Spartina alterniflora Loisel., a halophyte grass model to dissect salt stress tolerance
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- Subudhi, P.K. & Baisakh, N. In Vitro Cell.Dev.Biol.-Plant (2011) 47: 441. doi:10.1007/s11627-011-9361-8
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Salinity is one of the most serious abiotic stresses affecting crop productivity worldwide. Improving tolerance to salinity in field crops is globally important because a majority of the world population relies on salt-sensitive crops such as rice, corn, and wheat for their daily calories. Although there is no salt stress sensor yet identified, different signaling components and tolerance mechanisms have been substantiated to a great extent in a glycophyte like Arabidopsis, and more recently in a few halophytes. With the rapid advances in genetics, genomics, and biochemical and transformation tools, it is now possible to explore the genetic and molecular basis of the unusually high level of salt tolerance in halophilic plants. We will focus on a halophyte grass, Spartina alterniflora, commonly known as smooth cordgrass, which possesses all known mechanisms of salt tolerance and subsequent exploitation of its genome information for crop improvement. A number of candidate genes encoding transcription factors, ion transport, osmoprotectants, antioxidants, detoxifying enzymes, etc. have been identified. Although recent efforts to develop salt tolerant cultivars that could retain the halophytic traits through transgenesis show some promise, further exploration is needed to test the contribution of single or multiple salt stress-related genes or regulatory factors from halophilic plants, including S. alterniflora, for possible utilization in crop improvement.
KeywordsAbiotic stressCrop improvementGenomicsGrassIon homeostasisSalinitySmooth cordgrass
Salinity and World Food Security
Among abiotic stresses, salinity poses a major threat to the global food security (Munns 2002). More farm land is becoming unsuitable for farming all over the world every year because of improper drainage, sea water intrusion in coastal areas, irrigation with poor quality water, and salt accumulation in dry areas. Increased salinization will continue to be a major problem because of irrigation of drylands and deserts, which constitute 50% of the land surface of this planet. It is estimated that 1–2% of the irrigated areas all over the world is becoming unsuitable for farming each year because of salinization, and most affected areas are concentrated in the arid and semi-arid regions (Food and Agriculture Organization 2002) (http://www.fao.org/WorldFoodSummit/english/newsroom/focus/focus1.htm). Because there is little room for expansion of arable land, increasing productivity has become an important objective of plant scientists. Most crop plants have reached a plateau in productivity, and further increase may not be spectacular compared to earlier success. Increased productivity and yield stability must come from designing crop cultivars or hybrids with improved resistance to biotic and abiotic stresses. To ensure the food supply for every human being on this planet at the current level, food production will need to increase 57% by 2050 (Wild 2003). It is now widely acknowledged that plant growth and productivity will be severely challenged by the stresses resulting from extreme climatic patterns on a global scale, which warrants sustained and innovative research to stabilize crop productivity in stressed environments.
The problem of salinity on a global scale can be addressed through utilization of multiple strategies, such as the use of halophytic species with agricultural value, improved water management, and development of crop varieties with improved salt tolerance. Enhancing salt tolerance in major field crops will require harnessing the knowledge gained from genomic research. Particularly, understanding the molecular genetic basis of the salt adaptation mechanisms in halophytic plant species could provide clues to develop salt tolerant crops. This review will discuss the salt tolerant mechanisms in halophytes, including Spartina alterniflora, and the prospect of their utilization in crop improvement.
Ecological and Evolutionary Significance of S. alterniflora
S. alterniflora Loisel. (commonly known as smooth cordgrass) is used extensively for shoreline protection and tidal marsh restoration on the Atlantic and Gulf coasts of the USA. It is an effective soil stabilizer used on interior mudflats, dredge-fill sites, and other denuded sites in coastal marshy areas. It dominates the vast expanses of southern coastal salt marshes (Chabreck 1972) and plays an important role in estuarine ecology, both as a major producer and as an important agent for the redistribution of minerals in the sediments (Odum 1961; Williams and Mudroch 1969). Dense stands of smooth cordgrass along the shorelines are an effective barrier to both tidal surge and heavy winds during hurricane seasons. It filters heavy metals and toxic materials from the water columns (Kiesling et al. 1988).
S. alterniflora grows up to 4 ft tall, and its stems are hollow and hairless. The leaves lack auricles and have ligules representing a fringe of hairs. Flowers are borne on compressed spikes. It is a highly cross-pollinating species because of its protogynous condition. It is adapted to highly saline environments found in salt marshes, in which only the halophytic plants can survive. It can spread by seed, rhizome, or vegetative fragmentation (Daehler and Strong 1994). It can produce seeds in 3–4 mo under favorable conditions. Because seeds of S. alterniflora are short-lived (recalcitrant), seed banks do not exist for this species (Sayce and Mumford 1990). But it colonizes open areas rapidly because of its high growth rate and fast propagation of stems via rhizomes. It is a highly productive grass that uses the available nutrients efficiently and produces significant amounts of organic matter.
The genus Spartina represents a monophyletic lineage in the subfamily Chloridoideae of the grass family Poaceae (Hsiao et al. 1999). Most Spartina species, including S. alterniflora, originated from the New World (Mobberly 1956), but Western Europe is home to four species of Spartina: Spartina maritima, Spartina anglica, Spartina × townsendii, and Spartina × neyrautii. The habitat of Spartina is mostly in coastal intertidal zones, but some species may grow well in freshwater swamps, coastal dunes, and even drier prairies in central North America. Both polyploidy and aneuploidy are observed in Spartina genus with no known diploid species. The basic chromosome number of the Spartina genus is x = 10 and 2n = 40–124 (Marchant 1968). Flow cytometry analysis revealed that S. alterniflora has the biggest genome (3.6 pg or 1,763.9 Mbp) compared to Spartina cynosuroides (1.54 pg or 756.35 Mbp), Spartina patens (1.98 pg or 969.36 Mbp), and Spartina spartinae (2.0 pg or 979.78 Mbp) (Baisakh et al. 2009b).
S. alterniflora (2n = 4× = 62) is a rhizomatous, perennial warm season grass with the C4 pathway of photosynthesis. It is physiologically adapted to salt marsh habitat (Teal and Teal 1969). It is native to North America and was introduced to the west coast of the USA and the west European coast. In California and Washington, S. alterniflora became an aggressive invasive species. It hybridized with native Spartina foliosa on the California coast and formed an introgressed population of hybrids by repeated crossing and backcrossing, threatening the native S. foliosa population (Anttila et al. 1998; Sloop et al. 2009). In Europe, S. alterniflora hybridized with S. maritima (2n = 60), and the resultant sterile hybrid, S. × townsendii, produced a new fertile and invasive allopolyploid species, S. anglica (Ainouche et al. 2004). S. anglica populations displayed greater morphological plasticity to environmental fluctuations than its parents (Thompson 1991) and specifically enhanced physiological mechanisms under anoxic environment (Lee 2003). Increased fitness and adaptation observed in these newly formed species clearly indicate the evolutionary consequences of the allopolyploidization process that occurred 150 yr ago (Ainouche et al. 2003).
Genetic Variation for Salinity Tolerance in S. alterniflora
It is well known that wetlands plants vary in their tolerance to salinity stress, which leads to broad zonation of coastal vegetation such as fresh, intermediate, brackish, and salt marsh plant communities (Day et al. 1989; Bertness 1991). While S. alterniflora dominates coastal salt marshes, S. patens is the dominant plant in intermediate and brackish marshes (Duncan and Duncan 1987). Ecophenic (phenotypically plastic) variation in salt tolerance has been reported in S. alterniflora (Nestler 1977) and S. foliosa (Cain and Harvey 1983). Intraspecific variation in salinity tolerance has been investigated in S. patens (Silander 1979; Silander and Antonovics 1979; Pezeshki and DeLaune 1995; Seliskar 1995; Hester et al. 1996) and S. alterniflora (Pezeshki and DeLaune 1995; Seliskar 1995; Hester et al. 1998).
An evaluation of 25 clones of S. alterniflora by Hester et al. (1998) indicated that there is highly significant intraspecific variation in lethal salinity level (83–115 g/L). Despite many significant genotypic differences in morphological characteristics such as leaf rolling index, leaf expansion rates, above ground, below ground and total plant dry weight, and below ground to above ground biomass ratio prior to salinity stress, none of these characteristics was valuable in explaining salt tolerance (Hester et al. 1998). Therefore, anatomical and physiological/biochemical differences between S. alterniflora genotypes are probably more important in explaining genotypic differences in salt tolerance than are differences in morphology. However, significant intraspecific variation to lethal salinity level in S. patens was weakly correlated to variation in morphological attributes (Hester et al. 1996). Plant morphological traits could explain population difference in salt tolerance in glycophytes, but absence of such association in the case of halophytes (S. alterniflora) could be due to operation of specialized adaptation mechanisms like ion exclusion and salt secretion (Hester et al. 2001) because greater ion selectivity (lower leaf Na+/K+ ratio) was displayed by S. alterniflora but not by S. patens, a brackish water plant species or by Panicum hemitomum, a freshwater plant species. Pezeshki and DeLaune (1995) studied two populations of S. alterniflora from the Louisiana Gulf coast marshes and reported superiority of one population over the other in terms of net photosynthesis and better growth response under various salinity levels. The intraspecific genotypic and phenotypic variations in S. alterniflora should open up the possibility of identifying and developing salt tolerant genotypes combined with high productivity for marsh restoration (Seliskar 1995).
Molecular Marker and Tissue Culture Studies in S. alterniflora
The application of molecular markers in Spartina has been limited to genetic diversity and evolutionary studies. Few studies on genetic diversity utilized Random Amplified Polymorphic DNA (RAPD) (Stiller and Denton 1995; O’Brien and Freshwater 1999; Ryan et al. 2007) and Amplified Fragment Length Polymorphism (AFLP) markers (Travis et al. 2002; Perkins et al. 2002; Subudhi et al. 2008; Utomo et al. 2008). The study of Stiller and Denton (1995) suggested that all S. alterniflora clones in Willapa Bay are descended from a single genet. Genetic variability in S. alterniflora clones from the Atlantic and Gulf coasts was analyzed by O’Brien and Freshwater (1999), and the clones were grouped into three distinct clusters correlating to geographic regions. Travis et al. (2002) characterized genetic diversity in S. alterniflora from restored wetland and undisturbed wetland sites and concluded that genetic diversity in restored populations is comparable to natural populations. But Perkins et al. (2002), from a similar study using AFLP, reported that the marshes with imported planting materials were less diverse than the natural marshes. Genetic relationship among the S. alterniflora samples collected from the severely affected dead zones and lightly affected peripheral transition zones of brown marshes was compared with those from healthy marsh areas (Subudhi et al. 2008). They observed that the surviving brown marsh individuals are genetically different from those of healthy marshes, and their survival could be due to favorable combination of genes responsible for tolerance to multiple abiotic stresses.
Molecular markers have been exploited to determine the extent and degree of hybridization between S. alterniflora and S. foliosa, and identity of S. alterniflora ecotypes (Daehler and Strong 1997; Ayres et al. 1999; Daehler et al. 1999; Anttila et al. 2000). Using both RAPD and Inter-Simple Sequence Repeat (ISSR) markers, Ayres and Strong (2001) confirmed multiple times that the hybrid origin of S. × townsendii from the cross between S. alterniflora and S. maritima and accounted for its extensive genetic variation to the hybridization of S. alterniflora with different S. maritima plants.
Salmon et al. (2005) compared the AFLP and methylation-sensitive AFLP pattern of S. × townsendii, S. × neyrautii with its allopolyploid S. anglica, and the parents S. alterniflora and S. maritima to study the effect of hybridization and genome duplication on polyploid genome evolution and adaptation. A significant level of parental methylation pattern altered in hybrids and the allopolyploid indicated a high level of epigenetic regulation, which could be responsible for its morphological plasticity and adaptation to a wide range of habitats. In contrast, another study indicated rapid structural changes in the genome of S. anglica (Baumel et al. 2002). Recently, Baisakh et al. (2009a) suggested that gene-specific markers such as EST-derived Simple Sequence Repeats (ESSRs) derived from S. alterniflora were transferable across species of Spartina and could advance comparative genomic studies of Spartina species with different origin and environmental adaptation. ESSRs in combination with genomic SSRs (Blum et al. 2004; Sloop et al. 2005) could be used for genetic diversity analysis, genome mapping, and evolutionary studies in Spartina.
Tissue culture and plant regeneration protocols have been developed for S. alterniflora (Wang et al. 2003a), S. patens (Li et al. 1995), and S. cynosuroides (Li and Gallagher 1996). To utilize S. alterniflora as phytoremediater, Czako et al. (2006) produced transgenic lines with two genes—organomercurial lyase (mer B) and mercuric reductase (mer A)—by Agrobacterium transformation that are resistant to both organomercurials (e.g., phenyl mercuric acetate and mercuric chloride).
Salt Tolerance Mechanisms in S. alterniflora Vis-À-Vis Other Halophytes
Halophytes are plants that grow and survive in highly saline environments (Flowers and Colmer 2008). Some halophytes require saline conditions for optimal growth, but others, including S. alterniflora, grow optimally without salt. S. alterniflora is also noted for its exceptional tolerance to a wide range of environmental conditions: inundation up to 12 h a day, pH levels from 4.5 to 8.5, and salinity from 10 to 60 parts per thousand (Landin 1991).
The ability to tolerate high salinity in the case of S. alterniflora and other halophytes is due to the operation of several cellular, organizational, and whole plant adaptation mechanisms (Epstein 1980). A number of mechanisms at cellular level, such as production of compatible osmolytes, protection of cell wall integrity, enzyme sensitivity, and control of ion movement, contribute toward salinity tolerance. The accumulation of compatible solutes such as sugars, polyols, amino acids, and tertiary and quarternary ammonium and sulfonium compounds, synthesized in response to salt stress in the cytosol and organelles, balances the osmotic pressure of the ions and thus protects the subcellular structures and minimizes oxidative damage (Hasegawa et al. 2000). In both S. alterniflora and S. patens, proline accumulates in response to salt stress (Cavalieri and Huang 1979; Cavalieri 1983), whereas glycine betaine is accumulated in S. alterniflora (Cavalieri and Huang 1981). Dimethylsulphoniopropionate (DMSP) is believed to play a role in salt tolerance as a constitutive organic osmoticum in leaf blades of S. alterniflora, in contrast with the role played by other organic solutes like glycine betaine, proline, and asparagine (Colmer et al. 1996). Wyn-Jones et al. (1977) proposed a model for osmoregulation in which Na+ accumulates in the vacuole, and organic osmotica (primarily betaine and proline) accumulate in the cytoplasm of cells in shoot tissue of S. × townsendii.
In the short form of S. alterniflora, reduced growth might be due to failure of osmoregulation (Cavalieri and Huang 1981). Leaf expansion is inhibited because of the fall in leaf turgor pressure (Drake and Gallagher 1984), possibly because of reduced Na+ uptake or reduced accumulation of organic osmotic, or both, in response to an increase in interstitial water salinity (Bradford and Hsiao 1982). Naidoo et al. (1992) investigated the effect of water logging and salinity stressors on the anatomy and metabolism of both S. alterniflora and S. patens and concluded that both species are adapted to water logging and salinity, but S. alterniflora appears to be more tolerant of reducing soil conditions and less responsive to higher salinity than S. patens, and accumulation of proline in both roots and leaves is increased at high salinities in both S. alterniflora and S. patens.
Protection of cell wall integrity is important for cell growth during salt stress (Iraki et al. 1989). The study of Gettys et al. (1980) revealed that greater salt tolerance of S. alterniflora compared to S. patens may be partly due to the differential sensitivity of three enzymes—leucine aminopeptidase, peroxidase, and malate dehydrogenase—to salt in vitro in leaf extracts.
A major component of cellular salt tolerance in halophytes is the control of ion movement across tonoplast and plasma membrane to ensure maintenance of Na+ concentration within tolerable limits in the cytoplasm (Wyn-Jones and Gorham 2002). S. alterniflora exerts fairly dramatic control over ion accumulation through selective uptake of potassium and excluding sodium (Smart and Barko 1980; Bradley and Morris 1991). It has been estimated that ion exclusion in S. alterniflora accounts for more than 90% of the theoretical maximum ion uptake (Bradley and Morris 1991). Furthermore, specialized salt glands in S. alterniflora (Anderson 1974; Nestler 1977) capable of secreting approximately half of the ions (Bradley and Morris 1991) also help in adjusting to the changing osmotic environment. To avoid the toxic effects of salt buildup on its cells, its cell membranes actively exclude most types of salt (Teal and Teal 1969).
Ion movement across plasma membrane is facilitated by plasma membrane-H+ATPase (PM H+-ATPase), which generates electrochemical gradient (Sze 1985). Wu and Seliskar (1998) studied the activity of PM H+-ATPase in response to salinity in S. patens callus and reported that there is 2–3-fold increase in activity of this enzyme when intensity of salt stress increases. But the amount of H+ATPase on the plasma membrane did not change. It is also known that halophytes have greater ability than glycophytes to induce PM H+ATPase activity in response to salt stress (Niu et al. 1993). Vacuolar ATPase (V-ATPase) and vacuolar H+-pyrophosphatase (V-PPase) provide energy for ion transport across the tonoplast into vacuoles (Gaxiola et al. 2007). Vacuolar Na+/H+ antiporters take advantage of the proton gradient formed by these pumps to exchange sodium ions for hydrogen ions. In halophyte Salicornia bigelovii, protein level and activity of V-PPase were increased under salt stress imposed by 200 mM NaCl, resulting in vacuolar Na+ accumulation (Parks et al. 2002). V-ATPase activity is increased in response to salt stress in Mesembryanthemum crystallinum (Ratajczak et al. 1994), and Na+/H+ exchange activity in vesicles prepared from leaves was constitutively high and further increased under salt stress (Barkla et al. 2002). In Suaeda salsa, activity of V-ATPase and Na+/H+ antiporter was increased under salinity stress (Wang et al. 2001; Qiu et al. 2007). In Thellungiella halophila, a salt tolerant close relative of Arabidopsis, salt stress increased vacuolar Na+/H+ antiporter activity but not that of the plasma membrane (Vera-Estrella et al. 2005). These pieces of evidence suggest that efficient sequestration of Na+ into vacuole, which is aided by increased activity of Na+/H+ antiporter and proton pumps, plays a critical role in salt tolerance.
Succulence and salt excretion are the responses of halophytes at tissue level under salt stress. In halophytes, the effect of ion toxicity is diluted by increasing the plant succulence (Waisel 1972). In Sporobolus spicatus, sodium and chloride ions are selectively secreted (Ramadan 2001). Excess salts are removed in Salicornia, Halocnemum, and Allenrolfea by discarding salt-saturated fleshy cortex and leaves (Chapman 1968). In T. halophila, accumulation of Na+ was less, and K+/Na+ selectivity of root plasma membrane channels was high compared to Arabidopsis (Volkov et al. 2003). The stomatal response to salinity is also considered as a salt tolerance mechanism (Robinson et al. 1997). S. alterniflora seedlings have all of the anatomical features of the C4-Kranz syndrome. It excretes salt through hydathodes (Waisel 1972). Other anatomical features include infrequent occurrence of stomata on the adaxial surface of the lemma and not at all on the abaxial surface, resulting in efficient conservation of water (Walsh 1990). Photosynthesis rates in S. alterniflora are generally quite high, even at salinities close to open ocean water (Longstreth and Strain 1977).
Overall, the halophytes are better adapted to germinate under saline conditions. But the germination is severely reduced at salinity level above 250 mM NaCl (Malcolm et al. 2003). The maximum tolerance limit for germination in S. alterniflora is between 6% and 8% NaCl (Mooring et al. 1971). The ability of S. alterniflora to germinate and grow under external hypoxia and high salinity gives this species a competitive advantage over other species in low lying marsh environments (Wijte and Gallagher 1996). Increased salinity when coupled with prolonged drought reduces the ability to nutrient uptake and could result in large-scale browning of marshes (Brown et al. 2006). To date, definite mechanisms of salt tolerance at the germination stage have not been defined.
There is a difference between glycophytes and halophytes with respect to accumulation of ions in higher concentrations, particularly in the leaves (Flowers et al. 1977). The successful salt tolerance response in halophytes could be due to improved ion selectivity, superior regulation of transport and sequestration of ions in vacuoles, and effective coordination of such partitioning to allow growth under salinity stress. Halophytes rely on Na+ for lowering the osmotic potential in aerial portions of the plant, resulting in water uptake, transport, and lowering of the metabolic cost of producing osmolytes. Kant et al. (2006) suggested that differential gene expression between glycophytes and halophytes contributes to the salt tolerance of halophytes after comparing T. halophila and Arabidopsis thaliana.
Molecular Basis of Salt Stress Tolerance in S. alterniflora
We describe in this section the research done in our laboratory toward understanding the molecular basis of this grass halophyte’s tolerance to salt stress and establishing it as a model halophyte.
Differential expression of genes from S. alterniflora under salt stress.
Differential response of transcription factors and genes of unknown functions from S. alterniflora to salt stress.
The varying expression pattern and level of different TFs in leaf and root tissues under salt suggested that their expression was under developmental control, and one kind of stress may simultaneously activate many TFs. Induction of ADF in response to cold and drought stress in wheat and rice respectively has been reported (Ouellet et al. 2001; Ali and Komatsu 2006). A C2C2-type zinc finger protein upregulated the expression of Cu (Zn) oxide dismutase that is involved in detoxification of reactive oxygen species (ROS) during oxidative stress in rice and Arabidopsis (Kliebenstein et al. 1999; Wang et al. 2005). Various types of ZFP transcription factors were reported to improve abiotic stress tolerance (Winicov and Bastola 1999; Mukhopadhyay et al. 2004; Sakamoto et al. 2004).
Stress-inducible promoters from S. alterniflora.
Promoters, which are induced by different abiotic stresses, have been studied to some extent (Yamaguchi-Shinozaki and Shinozaki 1994), and activity of most of these inducible promoters is poor compared to constitutively expressed promoters (Holtorf et al. 1995). More stress-inducible promoters including their fine-tuning are essential to obtain desirable transgene expression to improve abiotic stress tolerance in plants. Particularly, in the exploitation of regulatory genes, stress-inducible promoters will be indispensable to improve abiotic stress tolerance using transgenic strategy. Toward this end, a promoter (1,875 bp) of a gene (SaAsr1), similar to rice abscisic acid (ABA) and osmotic stress-inducible gene, was isolated and characterized through genome walking (Baisakh et al., unpublished). The stress-inducible expression of this gene in leaves has been verified by reverse transcription PCR. SaAsr1 was upregulated in leaf rather than in root, and the expression was constitutive with no change under salt stress (data not shown). The functionality of the SaAsr1 promoter was confirmed by the transient expression of the gusA transgene under the full-length promoter in immature embryo-derived callus of rice variety “Cocodrie.” In silico analysis of the promoter revealed the transcription start site (TSS = +1) mapped to an adenine residue (A), which was 86 nt upstream of the start codon (ATG). The TATA box was located −25 bases upstream the TSS. A number of cis-regulatory motifs, viz., DRE-CRT (Dehydration Response Element/C-Repeat), ABRE (ABA-responsive element), CBF (C-repeat binding factors), LTRE (low-temperature-responsive element), ERE (Ethylene-responsive element), LRE (light-responsive element), etc., were present in the promoter. DRE is an essential element for regulating induction of many drought and cold-inducible genes, including rd29A (Yamaguchi-Shinozaki and Shinozaki 2005). Similarly, CRT and LTRE elements are known to regulate cold-inducible expression of genes. The cis-acting elements were characterized in transgenic Arabidopsis plants through deletion promoter/reporter gene (gus) fusion constructs. The analysis showed that a minimal promoter of 203 bp, along with two cis-acting elements (ABRE, ERE), is enough to confer abundant expression of the gus gene under abiotic stresses (salt, ABA, and PEG) (Baisakh et al., unpublished). This promoter was found to be unique in that it contains 1 or 2 cis-acting elements that specifically respond to salt stress as well as ABA and PEG (Baisakh et al., unpublished).
Similarly, in silico analysis of the promoter (1,295 bp) of a differentially expressing SaCTP gene identified the transcription start site (TSS = +1) to a cytosine residue (C), which is 34 nt upstream of the start codon (ATG). The TATA box was located −25 bases upstream of the TSS. There were four ABRE in addition to a number of cis-regulating binding motifs, viz., DRE-CRT, CBF, LTRE, E-BOX, and MYB (Baisakh et al., unpublished).
The promising data obtained so far are compelling to expand its genomic resources and establish this grass as a model to exploit its resources for improvement of salt tolerance, especially in cereal crops.
Genetic Engineering with Halophytic Genes to Improve Salt Tolerance
Transgenic salt tolerant plants developed with genes from halophytes
Ohta et al. 2002
Wu et al. 2009
Zhao et al. 2006a
Zhang et al. 2008
Li et al. 2008
Qiao et al. 2007
Ardie et al. 2009
Guo et al. 2006
Gao et al. 2006
Lv et al. 2008
Na+/H+ antiporter and H+-PPase
Zhao et al. 2006b
Shen et al. 2002
myo-Inositol O-methyl transferase
Sheveleva et al. 1997
myo-Inositol phosphate synthase (MIPS)
Das-Chatterjee et al. 2006
Baisakh et al. 2009a
Betaine aldehyde dehydrogenase (BADH)
Li et al. 2003b
Wu et al. 2008
Jia et al. 2002
Li et al. 2003a
Cacineurin B-like protein
Sun et al. 2008
Inositol polyphosphate kinase
Zhu et al. 2009
Mahalakshmi et al. 2006
Allene oxide cyclase
Yamada et al. 2002
Chen et al. 2007
Copper/zinc superoxide dismutase
Prashanth et al. 2008
Han et al. 2008
Metallothioneine and UDP-galactose epimerase
Endo et al. 2005
DREB1 (EREBP/AP2-type protein)
Shen et al. 2003
Unknown function gene (Bg70 and cyc02 homolog)
Ezawa and Tada 2009
Transgenic approaches to salt tolerance are mainly achieved through the overexpression of the genes involved in Na+ extrusion from the root or Na+ compartmentalization in the vacuoles (Wang et al. 2003b; Bhatnagar-Mathur et al. 2008; Uddin et al. 2008). Transgenic rice (cv. Kunihikari) expressing a Na+/H+ antiporter (AgNHX1) gene from Atriplex gmelini survived under salt stress (300 mM) for 3 d, while the wild-type (WT) plants could not (Ohta et al. 2002). Moreover, the transgenic rice plants grew normally, although with reduced tillers and only young leaves surviving, and set seeds after 3.5 mo. The increased activity of the Na+/H+ antiport into vacuoles is likely to enlarge the capacity for Na+ deposition in mature leaves. Such leaf-to-leaf compartmentalization that effectively controls the differential distribution of Na may improve the salt tolerance at cellular level and in turn may protect the younger leaves from salt toxicity (Munns 1993). Similarly, transgenic Arabidopsis overexpressing Na+/H+ antiporter (SsNHX1) from S. salsa grew normally and completed its life cycle unaffected under 200 mM NaCl, while the growth of WT plants were retarded (Li et al. 2007). The transgenic plants maintained slow growth but were greener than the WT plants. Although not statistically significant, more Na+ was accumulated in transgenics than the WT Arabidopsis. In another study, Li et al. (2009) observed that overexpression of SsNHX2 (an alternative splicing variant of SsNHX1) in Arabidopsis resulted in vigorous growth of transgenics with higher fresh and dry weights and Na+ and K+ accumulation compared with the WT plants. Overexpression of Na+/H+ antiporter gene (AlNHX1) from Aeluropus littoralis conferred on tobacco plants a tolerance to salt and maintained a high K+/Na+ ratio in the leaves compared with the WT plants. Ionic analysis indicated that under a month-long salt stress, the transgenics compartmentalized more Na+ in roots, and reduction of K+ uptake was much more than that in WT type plants (Zhang et al. 2008). Silencing of ThNHX1 gene from T. halophila resulted in salt sensitivity of the transgenic plants, whereas the seedling survival rate was doubled at 200 mM NaCl in overexpresser Arabidopsis plants compared to the WT plants (Wu et al. 2009). Furthermore, the transgenic plants flowered normally and had higher fresh and dry weight than those of WT plants, which wilted under salt. However, there was no significant difference between transgenic and WT plants with respect to Na+ and K+ ions. Transgenic overexpresser lines with NHX1 genes from a halophyte Chenopodium glaucum and glycophyte rice did not differ much with their salt tolerance phenotype (Li et al. 2008). Nevertheless, the transgenics showed enhanced salt tolerance by accumulating more Na+, higher dry weight, and greener leaves compared with the WT plants.
The Na+/H+ antiporters work with a proton-motive force generated by the vacuolar ATPase and pyrophsphatase (PPase). Theoretically, overexpression of H+-PPase should enhance the ability to form the electrochemical proton gradient between cytoplasm and vacuole to mediate Na+ sequestration into vacuoles by Na+ (or Ca2+)/H+ antiporters. Experimental evidence of such a hypothesis was provided by Gao et al. (2006), who reported that transgenic tobacco overexpressing ThH+-PPase gene from T. halophila accumulated 20–30% more Na+ in leaf tissues than the WT plants. The transgenic lines showed viability of mesophyll protoplasts and produced higher biomass than that of WT plants under salinity. Cotton plants transformed with ThH+-PPase also showed enhanced salt tolerance with improved photosynthetic performance in comparison to the WT plants (Lv et al. 2008). Heterologous expression of SsH+-PPase from S. salsa increased salt as well as drought tolerance of transgenic Arabidopsis (Guo et al. 2006). The transgenic plants could survive up to 200 mM NaCl with higher Na+ contents and better growth compared with the WT plants. A high-affinity K+ transporter gene PutHKT2;1 from Puccinelia tenuiflora resulted in higher Na+ contents in Arabidopsis than the WT by controlling the Na+ homeostasis (Ardie et al. 2009). The transgenic plants exhibited a significantly reduced K+ content in roots and leaves, suggesting that it acted as a negative regulator of K+ uptake system in transgenics.
Increased salinity tolerance of a range of plant species overexpressing NHX gene or PPase gene from the halophytes and non-halophytes indicates that these genes are involved in Na+ tolerance in plants. The differences in the expression levels of these two genes may affect the potential to sequester Na+ in vacuoles of the leaves. Although, expression of either NHX or H+-PPase gene conferred salinity tolerance in transgenic plants (Guo et al. 2006; Zhao et al. 2006a), co-expression of SsNHX1 from S. salsa and AVP1 from glycophyte Arabidopsis resulted in greater salt tolerance in transgenic rice compared with the single-gene transformants (Zhao et al. 2006b). In a different note, Bhatnagar-Mathur et al. (2008) suggested that a strategy of just Na+ excretion would appear inadequate for imparting salt tolerance in crop plants.
Protein kinases are involved in several signal transduction pathways and are implicated in different biotic and abiotic stresses. Overexpression of T. halophila CBL-9 homolog conferred salt tolerance in transgenic Arabidopsis (Sun et al. 2008). The transgenic lines had better seed germination than the WT after 3 d of salt stress up to 200 mM NaCl. In the later stage of salt stress, the WT plants suffered severe reduction in root growth, fresh and dry weight biomass, whereas the reduction was less in transgenic plants. Another kinase gene from T. halophila, ThIPK2 encoding inositol polyphosphate kinase, improved salt tolerance when overexpressed in Brassica napus (Zhu et al. 2009). While all WT plants died in 2 mo after exposure to 200 mM NaCl, the transgenic plants survived, and the growth was only slightly affected with a positive correspondence to the expression of ThIPK2 protein in the transgenics. The transgenics had greater biomass than the WT plants under 100 mM NaCl. Moreover, the K+ content in the roots and leaves of the transgenics was higher than the WT plants but with no obvious difference in the Na+ content between the two. A serine protein gene (PcSrp) from P. coarctata afforded salt tolerance when expressed in finger millet (Mahalakshmi et al. 2006). The transgenic millet showed significantly higher root length, shoot length, and seedling fresh weight after 1 mo of salt stress (250 mM NaCl). The total Na+ and K+ content was higher in the transgenic roots but lesser in shoots when compared to that of WT plants.
Antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), etc., play an important role in abiotic stress tolerance by detoxification of xenobiotics achieved through the quenching of the reactive oxygen molecules. A few transgenics have been developed with halophytic genes encoding antioxidant enzymes. Transgenic rice overexpressing the SsGST gene from S. salsa had reduced oxidative damage from salt stress (Zhao and Zhang 2006a). This was achieved by accumulation of less H2O2 and malon dialdehyde (MDA) in transgenics compared to the WT plants. Furthermore, the transgenics also showed a simultaneous increase of SOD and CAT activity along with the GST expression. Similar results were obtained when SsGST was co-expressed with SsCAT in rice (Zhao and Zhang 2006b). The transgenic rice had reduced relative electrolyte leakage in addition to reduced H2O2 and MDA contents. Although no agronomic data was presented, the transgenic rice showed tolerant phenotype under 200 mM NaCl stress in greenhouse experiments (Zhao and Zhang 2006b). A cytosolic copper/zinc superoxide dismutase (Cu/ZnSOD), from a mangrove plant, Avicennia marina conferred the transgenic overexpresser rice plants to tolerate 150 mM NaCl for a period of 8 d under hydroponics, whereas the WT plants wilted by the fourth day of the stress treatment (Prashanth et al. 2008). Under pot culture, the transgenics survived salt water (100 mM NaCl) irrigation for 15 d and produced up to 28% higher grain yield compared with the WT plants. Overexpression of a phytoene synthase gene involved in carotenoid biosynthesis pathway from Salicornia europaea manifested the salt tolerance phenotype of transgenic Arabidopsis via increased photosynthesis rate and the photosystem II activity. The transgenics had enhanced growth, increased SOD and peroxide activity, and reduced H2O2 and MDA activity (Han et al. 2008).
Conclusion and Future Perspectives
Although genetic basis of salinity tolerance is well established due to the presence of salt tolerant plants (halophytes) and genotypic variation in crop species (Epstein et al. 1980), development and release of fewer salt tolerant cultivars to date clearly demonstrate that there has been little progress in this regard (Flowers 2004). Since halophytes have a clear edge over glycophytes in adaptation to saline environment, halophytic models are essential to investigate the molecular basis of salt tolerance mechanisms. But it is not known if salt tolerance mechanisms in all halophytes are the same or there is any specific mechanism that has evolved in halophytes that is essential to respond favorably in diverse environments (Flowers and Colmer 2008). Superior ability of halophytes compared with glycophytes to survive salt shock allows the former to adapt and continue growth in saline environment (Hasegawa et al. 2000). Like halophytes, a few glycophytes possess some ability to respond to salt stress and adapt to a substantially higher level of salinity when stress imposition is gradual. Thus, it remains to be investigated if there are particular biochemical mechanisms which are better activated or preactivated to elicit quick and better response in halophytes resulting in adaptation to saline environment.
The Poaceae is the major group with 143 monocotyledonous halophytic species (Flowers et al. 1986). Because of wide range of habitat, halophytes may be using diversity of mechanisms, which can be clarified using different halophytic models (Flowers and Colmer 2008). Comparison of mechanisms in transport and regulatory mechanisms using different model organisms, including S. alterniflora, will be required to understand their ability to tolerate salt stress and the underlying mechanisms. Although the general response to salt stress is similar in all plants, halophytes have evolved unique mechanisms or regulatory pathways in sensing, transducing, and coordinating the adaptive mechanisms in response to salt stress (Bohnert and Cushman 2000; Wong et al. 2006). Particularly, the monocotyledonous halophytes should be given due attention because there are fundamental differences in development and anatomy and different traits associated with abiotic stress tolerance mechanisms between monocot and dicot plants, which make it difficult to apply the knowledge from dicot model species to improve salinity tolerance in major food cereals (Tester and Bacic 2005). There is a clear difference in the use of Na+ and K+ between mono- and dicotyledonous halophytes (Flowers and Colmer 2008). Dicotyledonous halophytes are more succulent than the monocots and thus accumulate more Na+ in their shoots than the monocotyledonous halophytes (Tester and Bacic 2005). There are a few reports on utilizing the genes from monocot halophytes (A. littoralis, P. coarctata, Aneurolepidium chinense, etc.) for crop improvement (Inada et al. 2005; Das-Chatterjee et al. 2006; Mahalakshmi et al. 2006; Shiro et al. 2007; Zhang et al. 2008), but progress achieved so far in monocots to understand the molecular basis of salt tolerance is not comparable to that achieved in dicot halophytes. This warrants finding a suitable monocot halophyte model to understand and exploit its salt tolerance mechanism (Flowers and Colmer 2008).
We propose S. alterniflora as a monocot halophyte model to investigate abiotic stress tolerance because of the following reasons:  It has all known salt tolerance mechanisms as described in earlier sections.  The comparative genetics approach is now proving effective in understanding the basic biology of the grasses and exploiting desirable genes from distantly related species (Devos and Gale 1997; Moore et al. 1995). The particular advantage of using S. alterniflora for salt tolerance improvement in crop plants lies in its high nucleotide/protein sequence similarity with that of the cereal food crops (Baisakh et al., 2006, 2008). S. alterniflora, being a grass species, will prove an ideal model plant species for understanding the evolution of halophytic adaptation as well as for subsequent translation of the information for improving salt stress tolerance in rice and other major food cereals (Flowers and Colmer 2008).  S. alterniflora maintains the state of stress anticipatory preparedness in non-stressful conditions and operates more specific regulation under salt stress.  Another positive feature of this plant is that several closely related species of Spartina, differing in their habitat preference and degree of stress tolerance, are available for comparative genomics studies. For example, S. alterniflora is tolerant to extreme salinity, high temperature and water logging, and it grows in low-lying high salinity marshes, whereas S. patens is adapted to high-lying brackish marshes, and S. pectinata grows favorably in non saline prairie areas. Flooding tolerance varies between S. alterniflora and S. patens due to the abundance and proliferation of aerenchyma tissues to oxygenate its roots and rhizosphere in the former and absence in later (Bertness 1991). Although in low oxygen environments, many plant species are unable to utilize nutrients in the substrate, S. alterniflora is efficient under such conditions because of rhizospheric oxidation. It has a large amount of aerenchymatous tissue extending from the leaves to the root tips, helping in gas exchange between the plant and the rhizosphere (Bertness 1991). Genetic variation in seed dormancy and recalcitrance, which are related to dehydration stress, is also prevalent in these species; S. alterniflora seeds are recalcitrant and dormant while S. pectinata seeds are orthodox and nondormant. Thus discovery of genes involved in salt tolerance mechanisms through EST or whole genome sequencing in this unexplored halophyte grass S. alterniflora and their comparison with those of closely related Spartina species will provide clues to understand the genetic basis of abiotic stress tolerance.  Multiple adaptations are required for the plants to survive in a saline environment. There is a need for specific genotypes of the crop plants, which are mainly grown in fresh water environments but occasionally inundated by the saline water. In that situation it is important to search for the genes that allow the plant to respond to salinity stress and maintain normal growth and developmental processes. The salt tolerant cultivars should be as productive as the other high-yielding cultivars in normal environments. S. alterniflora, being in this category, should provide some insights regarding this complex adaptation feature.
An important issue in using halophytic traits or genes in crop improvement is whether salt tolerance can be improved without compromising productivity. There have been numerous efforts to improve salt tolerance using the transgenic approach with a focus on ion homeostasis, osmolyte production, antioxidants protection, signaling and transcriptional regulation (Wang et al. 2003a; Yamaguchi and Blumwald 2005; Blumwald and Grover 2006). However, the non-correspondence of the performance of transgenics between controlled growth environments and natural field conditions is a major concern in developing products for commercial cultivation (Ashraf et al. 2008). Most of the studies involved single genes (Sahi et al. 2006). As mutigenic complexity of the stress response in field crops requires exploration of combined effect of several protective mechanisms (Bohnert et al. 2006), it is necessary to sort out the candidate loci and their superior alleles with significant contribution for salt tolerance and then pyramid them to target multiple mechanisms through multiple gene engineering or marker-assisted breeding (Rus et al. 2005). Thus, discovery of stress inducible promoters and genes involved in salt tolerance mechanisms needs to be explored in S. alterniflora and other halophytes for utilization in development of improved salt tolerant field crops.
Transcription factors that regulate functionally related genes could be particularly attractive targets to investigate their role in improving abiotic stress tolerance (Winicov 1998). Overexpression of some stress inducible TFs is suggested as a promising strategy to improve abiotic stress tolerance because of their ability to activate or repress genes through cis-acting sequences responding to specific stresses. Although there are a few reports on the manipulation of TFs (Kasuga et al. 1999; Kim et al. 2001; Park et al. 2001; Haake et al. 2002; Mukhopadhyay et al. 2004; Hu et al. 2006) to enhance abiotic stress tolerance in plants, such research from halophilic plants is scanty (Shen et al. 2003). Therefore, research efforts should be directed for utilization of TFs from halophytes, including monocots such as Spartina alterniflora for possible utilization in crop improvement (Cherian et al. 2006).
The financial support for this study from the United States Department of Agriculture-CSREES is gratefully acknowledged. This manuscript is approved for publication by the Director of Louisiana Agricultural Experiment Station, USA as manuscript number 2011-306-5590.