Isolation, identification and expression analysis of salt-induced genes in Suaeda maritima, a natural halophyte, using PCR-based suppression subtractive hybridization

Abiotic stresses are the principal cause of decreasing the average yield of major crops by more than 50% leading to losses worth hundreds of million dollars each year [1]. Among these, high soil salinity, contributed largely by Na⁺ and often compounded with drought, is the main factor that adversely limits the growth and productivity of the major crop plants, including rice. Nevertheless, plants do exist in nature, like the halophytes, which survive and grow under extreme of salinity; severe climate changes throughout millions of years have resulted in the evolution of flora that exhibit substantial genetic diversity for adaptation to environmental perturbations [2]. It is in fact also believed that the genetic diversity in glycophyte, particularly in the crop plants, has been narrowed down over the millennia because of loss of alleles contributing significantly to salt adaptability [2]. Hence, while there is a need to understand the plants' response to salt stress, and the salt tolerance mechanism itself, with the common aim of enhancing salt tolerance in the crop plants, it is necessary that such attempt should include preferably the halophytic species. This is required, as variation in salt tolerance in the crop plants is relatively small, although working with the crop species has direct implication for agriculture.

Decades of research on the effect of salinity on growth and development of various plants and their response to salinity treatment at the physiological and biochemical levels has generated a wealth of information on the salt tolerance related parameters or salt tolerance determinants in plants. These may be grouped into 1) morphology adaptation, reflected as thickening of the leaves and cuticular wax deposition [3], 2) osmotic adjustment, reflected as accumulation of compatible solutes in the cytoplasm [4], 3) maintenance of ion homeostasis, reflected as H⁺-pump functioning [5], K⁺/Na⁺ selectivity [6] and Na⁺ exclusion and compartmentation [7–9], 4) cell signalling and gene expression, reflected as abscisic acid (ABA) and jasmonic acid (JA) accumulation [10,11], regulation of salt overly sensitive gene-1, SOS1 [12, 13], Ca²⁺-induced increase in K⁺/Na⁺ selectivity [14], increase in CDPK (Ca²⁺-dependent protein kinase) and MAPK (mitogen-activated protein kinase) activities [15, 16] and synthesis of many transcription factors [15, 17–19], 5) oxidative stress mitigation, reflected as activation of the antioxidative machinery [20, 21], and 6) molecular trafficking and cell stability, reflected as the accumulation of heat-shock proteins (HSPs), jasmonic acid-induced proteins (JAIPs) and late embryogenesis abundant (LEA) proteins [15, 17, 22–25]. Although transgenic plants have been developed for many genes upregulated under salt stress, such as P5CS (Δ¹-pyrroline-5-carboxylate synthetase), DNA helicase, carbonic anhydrase (CA), glyceraldehydes-3-phosphate dehydrogenase, Na⁺/H⁺ antiporter [26–30], and the plants show enhanced tolerance to salinity, the field trials of many of them have remained highly unsuccessful [31]. Hence, the basics of salt tolerance still remain illusive, and needs further investigation.

The plant stress adaptive responses include dynamic transcriptome changes, presumably playing important role in co-ordination of the many different molecular events responsible for cellular and organismal homeostasis. These changes are generally regulated by complex signalling pathways, which are activated in response to various abiotic and biotic stimuli allowing the plants to cope with the changing environmental conditions [32]. There also occurs crosstalk between different signalling pathways [33, 34], and identification of the convergent and divergent pathways between salinity and other abiotic stress responses and the nodes of signalling convergence may greatly enhance the understanding of the salinity stress response and the salt tolerance mechanism. Although several studies have been carried out on abiotic stress responsive signal pathways [15, 35–37], and several reports exist on massive changes in the profile of gene expression in plants [38, 39], these are mostly on Arabidopsis or other glycophytes, which are sensitive to salt. Such studies on the native flora of saline environment, i.e. halophytes, are scarce, although better information on the salt tolerance determinants is likely to come from the work on these plants rather than the work on the glycophytes.

One of the techniques being largely used to identify stress-responsive genes is subtractive hybridization. Attempts have been made to identify the salt stress regulated genes by suppression subtractive hybridization (SSH) in rice [19] and tomato [18]. However, to the best of our knowledge, the technique has so far not been used to identify the genes differentially expressing under salt stress in salt-tolerant plants. Among the salt-tolerant photosynthetic organisms, nevertheless, the salt-stress upregulated ESTs have been cloned in an alga, Dunaliella salina [40]. However, only a few highly upregulated ESTs were sequenced for further studies. Hence, the present work was carried out with the aim of generating cDNA library of salt-induced genes in S. maritima, a natural halophyte, following PCR based SSH in order to get information on salt stress response in the plant at the transcript level. Moreover, most of the salt-stress upregulated ESTs were identified so as to get a comprehensive picture of the salt-stress response in the plant at the level of gene, which might be useful in elucidating the molecular mechanism underlying salt tolerance.

Over the last decade, vast insight into the plant growth and development and other plant processes have been gained because of the growth and development in molecular biology techniques. Suppression subtractive hybridization (SSH) is among such techniques being largely used to isolate the genes that are differentially expressed in contrasting environments. Although the PCR-based SSH technique has been used to know the genes differentially expressed in some plants under salt stress [18, 19, 40, 49], no report exists on the salt-responsive genes in natural halophyte, which is likely to give better information on the genes relevant to salt tolerance than the studies carried out on other plants. The present study has been an attempt in this direction. Although SSH is a powerful technique that enriches the differentially expressed genes, it is by no means perfect. This is also evident from the present study as the probe prepared using the reverse subtracted SSH cDNA showed hybridization with the forward subtracted SSH cDNA clones, or vice-versa (Fig. 1b, c), which was not expected. Northern blot analysis of select forward subtracted cDNA clones, including that showing hybridization with the radiolabelled reverse subtracted SSH cDNA, nevertheless, revealed that these clones (ESTs) actually represented the genes overexpressing in response to the NaCl treatment (Fig. 3). The Northern blot hybridization also revealed that the SSH process was in fact dependent on the expression level of a gene, as the genes showing high ESTs redundancy showed greater hybridization signal than those showing low ESTs redundancy (Fig. 3, Table 2). Expression analysis of a few genes by qRT-PCR also confirmed the same; the NaCl-induced changes in the transcript levels of the selected genes (Fig. 4) quite paralleled their ESTs redundancy (Table 2 and 3). Besides, the qRT-PCR analysis also revealed that although the ESTs of the genes like P5CS and Cat were present in very low redundancy, their expression in response to the NaCl treatment had in fact increased by more than four fold. Hence, the genes showing low overexpression in response to a stress application may not find representation in the forward SSH cDNA library. It is also possible that the cDNAs of certain gene, present in high amount in both the 'Driver' and the 'Tester' cDNA population, may not be subtracted properly from the 'Tester' cDNA population in two rounds of hybridization with the 'Driver' cDNA, and thus the gene may find representation in the forward SSH cDNA library without actually overexpressing in response to a stress application [19].

The overexpression of as many as 167 unigenes while suggested involvement of a large number of genes in the salt tolerance process, contig EST redundancy of 81.8% indicated the possibility of discovery of more such genes, particularly that of the low abundance proteins, on continued cloning and sequencing of the forward SSH cDNA library. Moreover, more than 30% of the unigenes were novel, not reported before, and in addition approximately 4% were found to be producing proteins of unknown function (Table 1, Fig. 6, 7). These transcripts might represent important genes specific to salt tolerance. Although the EST redundancy of the genes in these groups is not high (Fig. 7), their importance in salt tolerance processes cannot be ignored (Fig. 7). The nucleotide and the corresponding amino-acid sequence data revealed that several clones isolated in this study, marked '^b' in Table 2 and 3, and in the supplementary table (see Additional file 1), were significantly homologous to the salt stress-regulated genes/proteins reported for various plant species. Other proteins are not reported to be salt-induced.

The PCR-SSH has revealed overexpression of as many as twenty four transcription factors in tomato in response to NaCl-stress [18]. Using the same technique, Sahi et al. [19], however, has reported differential expression of genes of only two transcription factors, EREBP (ethylene responsive element binding protein) and Zn-finger (ZnF) protein, in two rice varieties in response to salt treatment. Our experiment on the other hand shows overexpression of the genes of six transcription factors in the test plant in response to the NaCl treatment (Table 2). The variation obtained could be species dependent. Nonetheless, the overexpression of the genes encoding EREBP and ZnF transcription factors are common among the three studies, suggesting important role of these proteins in the salt tolerance processes. EREBPs, currently known as ERE binding factor (ERF) proteins [50] belongs to a family of plant specific transcription factors characterized by the presence of ~60 amino acid highly conserved ERF/AP2 (APETLA2) DNA binding domain. A number of genes, including those encoding pathogenesis-related and antifungal proteins, are induced by various forms of biotic and abiotic stresses, such as pathogen attack, wounding, UV radiation, high or low temperature, drought and NaCl [50, 51] mediated by ethylene produced in response to these stresses [51]. In addition, many of these have been found to contain ethylene responsive element (ERE), a cis acting element identified as GCC box for the interaction with ERF [52]. Certain Arabidopsis ERFs have also been reported to be induced by abiotic stresses, such as salinity, independent of ethylene signal transduction [50]. Enhanced expression of S-adenosyl-L-methionine synthase (SAMS) in the present case although indicated ethylene synthesis (Fig. 2) and ethylene dependent accumulation of ERF protein, the accumulation of ERF protein was most likely independent of the ethylene signal transduction. This is because there occurred no enhancement in the expression of ACC synthase (S-adenosyl-L-methionine methylthioadenosine-lyase) gene required for the conversion of S-adenosylmethionine to ACC (1-aminocycloropane-1-carboxylic acid), a rate limiting step in ethylene synthesis [53]. The increase in SAMS could be required to take care of the requirement of S-adenosylmethionine (SAM) for other biochemical reactions as the compound is the major methyl donor in plants and is used as a substrate for many biochemical pathways [54], involved in methylation reactions that modify lipids, proteins, and nucleic acids [53].

As for ethylene, no genes were identified in the forward SSH cDNA library that could be involved in the synthesis of jasmonic acid (JA) or methyl jasmonate (MeJA) from linolenic acid [55]. This is despite the fact that the forward SSH cDNA library showed the presence of ESTs for two isoforms of the gene encoding JAIP with a combined redundancy of as high as 10.49% (Table 1, Fig. 3, 4). Besides being induced by JA, a few JAIPs have also been reported to be induced by drought and salt [56, 57]. The promoter of none of the genes encoding JAIPs has so far been studied. However, a jasmonate (and elicitor) responsive cis element (JERE) containing a GCC motif has been identified in the terpenoid involved alkaloid (TIA) biosynthetic gene strictosidine synthase, Str [58], which is recognised by a AP2 domain containing transcription factor ORCA2 (Octadecanoid-responsive Catharanthes AP2), similar to ERF, but its synthesis is induced by MeJa as elicitor instead of ethylene [58]. The involvement of the AP2-domain family members in both ethylene and JA signalling suggest that ethylene and JA may crosstalk via these transcription factors. Moreover, recently transcription factors like JERF1 (Jasmonate and ethylene response factor 1) and Tsil1 (Tobacco stress-induced gene 1) induced by NaCl, ethylene and JA have been discovered [49, 59]. Besides binding to GCC box, these transcription factors also bind to dehydration responsive element (DRE)/C-repeat (CRT) involved in drought, salt and cold stress responses [60]. This expands the horizon of the crosstalk not only between ethylene and JA, but also among the other abiotic stresses, dependent or independent on ethylene/JA signalling for biological response.

The homeodomain zipper (HDZip) genes, ATB1 and HDZ3, were among the highest expressed transcription factors, which is not only reflected from their ESTs redundancy (Table 2), but also from the Northern blot result (Fig. 3). The combination of a homeodomain and a leucine zipper motif is unique to plant kingdom, suggesting that the HDZip genes may be involved in regulation of developmental processes specific to plants [61]. The functional information available on HDZip genes suggest that at least some of these genes are involved in mediating the effect of external conditions to regulate plant growth and development [62]. Several A. thaliana HDZip genes, like ATHB-6, -7 and -12 have been reported to be involved in abscisic acid (ABA) related response, including water deficit [63, 64]. Several others, like ATHB-7, -12, -6, -21, -40 and -53, have also been reported to be overexpressed upon NaCl treatment, besides ABA treatment, particularly ATHB7 and -12, which showed 12 to 25 times upregulation [65]. However, ATHB1, to which the present HDZip finds maximum homology, have been found to be down regulated upon NaCl treatment [62]. The response of a member of a family of genes to the external environment could be, however, a species-specific phenomenon as CPHB-6 and CPHB-7 (Craterostigma plantagineum HDZip genes) upregulated upon ABA treatment [66], also finds maximum homology with ATHB-1. Hence, the HDZip genes overexpressed in the present case with a combined ESTs redundancy of 7.52% could be very important from the point of view of salt tolerance in plants.

The role of the other transcription factors in salt tolerance processes may not be ruled out as the genes of at least two more of them, C2H2 zinc finger (C2H2-ZnF) and white collar (WC1), were overexpressed (Table 2). Among them, the C2H2 type zinc finger protein with 176 members in A. thaliana [67] and 189 members in O. sativa [68], constitute one of the largest families of transcriptional regulators in plants. These are mostly plant specific, and synthesis of many of them has been found to be enhanced under salt [69–71] and other environmental stresses [70, 71]. The importance of the protein in salt tolerance is also substantiated from the fact that tobacco plant transgene for C2H2-ZnF (ZFP182, overexpressing in rice under salt stress) showed increased tolerance to salt stress [69]. With regard to WC-1, however, no report is available so far indicating its possible involvement in salt or abiotic stress tolerance. The protein is only known to mediate blue-light and circadian response [72]. The gene has so far not been found to be overexpressing under salt or other environmental stresses. Besides, the overexpression of the gene in Neurospora crassa does not result in any upregulation of the genes reportedly involved in salt or abiotic stress tolerance [72]. There is also no report of any abiotic factor accelerating accumulation of pasticcino-1 (PSA1), which regulates the function of NAC-like transcription factors by controlling its targeting to nucleus [73]. However, the NAC family of transcription factors, which is one of the largest transcription factor families in plant genomes, have not only been implicated in plant development [73, 74], but also in various abiotic stress responses [75]. Hence, it is plausible that PSA1 might be important from the point of view of salt tolerance depicting that a lots of cellular changes might be necessary for a plant to grow and perform under salt stress.

Overexpression of the genes encoding protein with various functional domains, such as CBS, F Box, C2 and C3H4 (Table 2) mediating important biochemical processes, mainly protein modification, degradation and membrane trafficking of proteins, is suggestive of their important role in adaptation of cells to NaCl enriched environment. In this regard, peroxin (PEX), a protein containing C3H4 Zn-RING finger, has been found to be involved in biogenesis of peroxisomes [76] important for not only carrying out fatty acid β-oxidation for energy generation, but also for protecting photo-damage of the photosynthetic machinery by carrying out photo-respiration. Besides, the organelle also harbour an antioxidant enzyme, catalase, required for eliminating H₂O₂ generated in plants under metabolic stress induced by NaCl [20], which otherwise would lead to oxidative damage to the cells. A RING finger containing protein (Rbx1) also forms a part of ubiquitin-proteosome system responsible for degrading the regulatory and misfolded proteins [77]; Rbx1 mediates binding of ubiquitin carrier protein (E2) to the multi-subunit ligase (E3) comprising of Skp1, culling1 and F-box protein (SCF), and the F-box subunit of E3 then recruits the protein to be poly-ubiquitinated and subsequently degraded [77, 78]. Overexpression of the genes encoding proteins with C3H4 and F-box motif in the plant in response to NaCl stress thus indicated enhanced synthesis of regulatory proteins, which are possibly destined to be degraded after their role in the adaptive processes are over.

The precise function of CBS (Cystathione-β-synthase) domain protein is yet to be understood, although thought to be regulatory. Overexpression of the gene encoding CBS domain containing protein and its presence in AMP activated protein kinase (AMPK), the cellular energy sensor, nevertheless, does suggest that salt adaptation could be linked to energy metabolism. In fact, it has been reported that CBS domain in AMPK has greater affinity for AMP than for ATP, and as the cellular energy content drops (low ATP, high AMP), binding of AMP to CBS domain of AMPK facilitates its phosphorylation making the enzyme active [79]. Once activated, AMPK drives the metabolic pathway towards ATP accumulation [79]. Besides, CBS domain is also present in plants in various chloride channels, which open upon binding of ATP to the domain, and thus it could be important from the point of view of regulation of membrane potential, Cl^- homeostasis and osmotic adjustment in plants under NaCl stress [80].

The overexpression of the genes encoding various transcription factors under NaCl stress in S. maritima is no doubt suggestive of great metabolic changes that might be occurring in plants depending upon their need for survival and growth under salt stress. However, these changes are not possible until the stress signal is perceived. This is supported in part by the overexpression of the gene encoding protein with C2 domain, a Ca²⁺ binding motif. Besides having affinity for Ca²⁺, C2 domain also displays remarkable property of recruiting a variety of other ligands and substrates, such as phospholipids and inositol phosphate [81]. Multiple copies of C2 domains have been identified in a growing number of eukaryotic signalling proteins that interact with cellular membranes and mediate a broad array of critical processes, including membrane trafficking, activation of GTPase for vesicular trafficking, control of protein phosphorylation and generation of lipid second messenger involved in signal transduction [81, 82]. The Ca²⁺-dependent tolerance of plants to NaCl [20, 83] could in fact be a result of enhanced synthesis of Ca²⁺-binding domain containing proteins. However, no known Ca-binding proteins, like Ca²⁺/calmodulin dependent protein kinase PsCCaMK, the Arabidopsis protein AtPC1, the membrane associated protein in rice OsEFA27 and Arabidopsis RD20, etc. was found to be overexpressed in the present case. The stress signal perception might also be G-protein mediated as overexpression of the gene encoding the protein (Transducin) was observed under NaCl stress in the present case; the involvement of G-protein in transduction of environmental signal is well documented [84]. However, none of the effectors in G-protein signalling was found to be overexpressed. The appearance of various phosphatases and kinases (Table 2, see Additional file 1), nevertheless, does suggest that many changes in the metabolic processes in response to external or internal signals must be mediated by protein phosphorylation and dephosphorylation.

Besides phosphorylation, O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins could be abundant in S. maritima under NaCl stress, as it appears from the overexpression of O-GlcNAc transferase (OGT) gene (Table 2), and hence this could be an important biochemical event in the salt tolerance process. A large number of nuclear and cytosolic proteins are O-GlcNAc modified, and has been reported to affect stability of proteins and their sub-cellular localization [85]. One mechanism by which O-GlcNAc addition affect the changes in protein activity is through competition between O-GlcNAcylation and phosphorylation for the modification of serine/threonine residues. In fact, reciprocal phosphorylation/O-GlcNAcylation of specific amino acid has been demonstrated for several proteins, including the transcription factor, c-myc, and the reciprocal modification was found to differentially affect the activities of these proteins [86]. However, not all the substrate proteins are regulated via reciprocal phosphorylation/O-GlcNAcylation. In some cases, O-GlcNAc addition may directly affect the protein activity [87]. Although there is no report of OGT overexpression under any environmental stress, OGT activity has been found to be essential for plant survival [87].

The salt adaptive metabolic changes could be mediated by the heat shock protein HSP70, a well known molecular chaperon. This is reflected from the overexpression of the genes encoding Bcl2 binding BAG and DnaJ proteins (Table 2), which physically interact with HSP70 [88, 89]. DnaJ like proteins are involved in a variety of processes including protein folding, protein partitioning into organelles, signal transduction and targeted protein degradation. Moreover, the DnaJ domain of the protein has especially been shown to interact directly with HSP70, thereby regulating its ATPase activity, which affects protein binding and folding [89]. Similar to DnaJ protein, BAG protein also has a conserved domain (BAG domain) to interact with the heat shock protein (HSP70/HSC70). Hence, the BAG protein might also be involved in protein folding and maturation [90]. The increased synthesis of BAG protein protects various cell types from heat-induced apoptosis, possibly through interaction with HSP70 and HSP40 [91].

In addition to the post translational events, pre-translational processes like mRNA and tRNA processing, and the translational event itself appear to be greatly changed or adjusted to suit the requirement demanded by salt adaptive physiological processes. This is evident from the significant increase in pre-mRNA splicing factor, 60S ribosomal P0 protein, appr-1p processing enzyme family protein, eukaryotic elongation factor 1A, translation initiation factor 2B-β sub-unit and valyl-tRNA. However, little is known about the role of these proteins in salt adaptation, or abiotic stress adaptation in general.

At the physiological level, the NaCl adaptive response was highly visible in terms of overexpression of the genes of many enzymes related to the synthesis and accumulation of glycinebetaine (Table 3). The most important among them being PEAMT mediating the conversion of phosphoethanolamine to phosphocholine, which is either dephosphorylated to form choline directly [92] or first incorporated into phosphatidylcholine and then metabolized to choline [93] (Fig. 2). Primarily the synthesis of choline occurs following the route phospho-ethanolamine (P-EA) to phospho-choline, P-choline (bold arrows, Fig. 2). However, the route P-EA to phosphatidylcholine, Ptd-choline (normal arrow, Fig. 2) also contributes substantially to choline synthesis depending upon the species [94]. The synthesis of the compound by other routes (broken arrows, Fig. 2) is also possible [94]. The choline produced in the cytoplasm is transported to the chloroplast where it is converted to glycinebetaine by the reactions catalyzed sequentially by choline monooxygenase (CMO) and betainealdehyde dehydrogenase (BADH). Thus, PEAMT is although not directly involved in the synthesis of glycinebetaine, the enzyme appears to be very important in the biochemical pathways of synthesis of the osmoticum. The activity of PEAMT has been reported earlier to be greatly enhanced in the betaine accumulating halophyte Atriplex nummularia [95] and glycophyte spinach [92] by salt stress. These, together with the overexpression of PEAMT in the present case with high ESTs redundancy (Table 3) suggest that increased synthesis of choline could be highly essential for the survival of plants under salt stress, particularly those accumulating glycinebetaine, and that S. maritima might be a glycinebetaine accumulating halophyte. However, no overexpression of the gene encoding BADH, the enzyme catalysing conversion of betainealdehyde to glycinebetaine (Fig. 2), the final step of glycinebetaine synthesis, was seen in the plant in response to the NaCl treatment (Fig. 4), although this has been reported for terrestrial glycophyte as well as halophyte [96, 97]. This could be because the availability of choline is probably more important for the accumulation of glycinebetaine than the amount of the enzymes catalysing the conversion of choline to glycinebetaine, i.e. choline monoxygenase (CMO) and BADH (Fig. 2). The fact is substantiated from the observation that the supply of exogenous choline leads to glycinebetaine synthesis even in the plants not accumulating glycinebetaine naturally, like Arabidopsis thaliana, Brassica napus and Nicotiana tobacum [98]. Moreover, modelling of the labelling kinetics of choline metabolites upon supply of ¹⁴C-choline demonstrated that choline import into chloroplast indeed limited its flux to glycinebetaine [99]. Hence, it was postulated that a high-activity choline transporter in the chloroplast envelope could be an integral part of glycinebetaine synthesis pathway in the species that accumulate the compound naturally [99]. The overexpression of three isoforms of choline transporter gene, each with high EST redundancy, in the present study appears to support the hypothesis.

The fact that choline synthesis is really enhanced upon salt treatment is supported from the enhancement in the expression of the gene encoding SAM synthesizing enzyme, S-adenosylmethionine synthase, SAMS (Table 3, Fig. 2) , which uses methionine and ATP as substrates. SAM is consumed in the glycinebetaine synthesis pathway for SAM-dependent methylation of ethanolamine (EA) or phosphoethanolamine (P-EA) in successive steps to produce choline, phosphocholine or phosphatidylcholine (Fig. 2). Besides, SAM is an essential substance for the living cells as a methyl group donor and as a precursor in ethylene biosynthesis catalyzed by ACC synthase and ACC oxidase (Fig. 2) [53, 100]. Hence, maintaining a considerable pool of SAM by enhancing the rate of its synthesis must be essential when the physiological condition so demand, as in the case of glycinebetaine accumulation under salt stress. In fact, it has been observed that in halophyte Atriplex nummularia accumulating glycinebetaine under salt stress, the transcript levels of SAMS co-regulates with that of PEAMT in response to varying salinity level [95]. The present work thus indirectly suggests that while going for the development of transgenic plant for enhanced accumulation of glycinebetaine, the attention should be focused on increasing the level of choline and its transport to chloroplast. Attention should also be paid to the fact that the transfer of the methyl group from SAM generates S-adenosyl-L-homocysteine (SAH), which is a potent inhibitor of SAM dependent methyltransferases. Hence, SAH should be hydrolysed or removed, and this is done by S-adenosylhomocysteine hydrolase (SAHH), breaking it into homocysteine and adenosine [101]. The overexpression of SAHH in S. maritima under salt stress in the present case is an indication that the plants accumulating glycinebetaine should overcome SAH accumulation, and that the plants transgenic for enhanced production of choline should also show enhanced expression of SAHH.

Besides glycinebetaine, proline is another osmoticum widely reported to accumulate in plants under salt stress. However, the report of accumulation of both glycinebetaine and proline in a plant in response to salt stress is limited [102]. The overexpression of the gene encoding P5CS (Table 3), the enzyme catalysing the conversion of Δ¹-pyrroline-5-carboxylate to proline, the final step in the conversion of glutamate to proline, nevertheless, does suggest that proline, in addition to glycinebetaine, might be accumulating in the plant under NaCl-stress. This may in fact be the requirement as the accumulation of glycinebetaine remains restricted to the chloroplast (Fig. 2), and hence the osmotic adjustment of the cytosol might be achieved by the accumulation of proline. Significant increase in the activity of P5CS (Fig. 5a), besides the expression of its gene (Fig. 3, 4), also indicated possible accumulation of proline in the plant in addition to glycinebetaine upon salt treatment.

Although the maintenance of cellular ionic homeostasis has been emphasized for the survival of organism, especially under ionic stress [7], no overexpression of the genes of any known cation transporters, particularly of the alkali cations, was observed in the present study, except of a putative Na⁺/H⁺ antiporter of low E value (Table 3). The finding is in contrast to the report of overexpression of Na⁺/H⁺ antiporter gene in several plant species under salt stress [7, 8]. Moreover, no Ca-binding protein or protein kinase was identified in the present study, in contrast to the SSH study in tomato [18], suggesting the absence of the SOS (salt overly sensitive) signalling pathway of Na⁺ efflux in the halophytes like S. maritima. Highly enhanced expression of the genes encoding at least two proteins (FC932784 and FG228211) finding high homology with the proteins conceptually translated as cation-efflux transporters from A. thaliana genome database, nevertheless, does suggest important role of cation efflux in salt tolerance, although the ion(s) they transport remains to be identified.

Several genes having no known relationship with salt tolerance were found to be overexpressing in the plant in response to the salt treatment. The two well known among them are that encoding CCL (CCR-like, cold circadian rhythm-RNA binding like) protein and carbonic anhydrase (CA). CCL gene encodes highly unstable mRNA, the stability being regulated by circadian clock [103]. The transcript of this gene is significantly more stable in the morning than in the afternoon [103]. However, the EST redundancy of the CCL gene (Table 3) in the present study indicated high accumulation of transcripts of the gene even in the evening (the plants for the isolation of RNA were harvested in the evening). Hence, it appears that the salt treatment either had increased the stability of the CCL transcripts or had enhanced the expression of the gene in the plant. Although the role of the RNA binding proteins in posttranscriptional regulation of gene function, critical for eukaryotic growth and development, is well documented [104], expression of none of the genes encoding these proteins, including CCL, has been reported to be affected by salt treatment. The physiological function of carbonic anhydrase on the other hand is well known, facilitating CO₂ availability for photosynthesis in C₄ and submerged aquatic plants [105–107]. The expression of its gene has also been reported to be highly enhanced in plants in response to salt treatment [29]. Besides, Arabidopsis plant transgenic for rice carbonic anhydrase (OsCA1) has been demonstrated to show greater salt tolerance than the wild type at the seedling stage [29]. However, any physiological or biochemical role of the enzyme in salt tolerance is yet to be established, especially in the non-aquatic angiosperm where the availability of CO₂ is not influenced by salinity. The tolerance of Dunaliella salina, a unicellular alga, to nearly saturating NaCl concentration, nonetheless, has been suggested to be in part due to increased accumulation of a halophilic plasma membrane CA isoform. The enzyme shows maximum activity at much higher NaCl concentration and is much more resistant to inhibition by salt than the enzyme isolated from the salt-sensitive alga Chlamydomonas reinhardtii; the unique characteristics of D. salina carbonic anhydrase potentially enable the enzyme to optimise inorganic carbon utilization in high salinities [105].

Xyloglucan endotransglycosylase/hydrolase (XTH) and expansin-3, both involved in cell wall metabolism, are also among the genes that have no biochemically or physiologically known relationship with salt tolerance, but were overexpressed upon salt treatment of the plant in this study (Table 3). One of them, XTH, a glucan endo-1,3-β-glucosydase, has also been reported to be greatly overexpressed in tomato upon salt treatment [18]. XTH catalyses endo cleavage of xyloglucan polymers and subsequent transfer of the newly generated reducing ends to other polymeric or oligomeric xyloglucan molecules and thereby participates in cell wall formation and elongation [108]. Expansin-3 on the other hand belongs to a group of extracellular non-enzymatic cell wall protein, which loosens the linkage between cellulose microfibrils by modifying the cell wall matrix in terms of increasing the mobility of the constituent matrix polymers [109]. The modification allows the cell wall to yield to the tensile stress created in the wall by the turgor pressure. Enhanced expression of XTH and expansin-3 in the present study seems to be in agreement with the visibly healthy growth and flaccid leaves of the plant grown in the saline medium than that grown without salt. However, no gene encoding expansin has so far been reported to be overexpressing in response to salinity. The maintenance of a greater leaf turgidity in the plant grown on salt than that grown without salt could be by accumulation of osmolytes, as discussed above.

An important physiological event that is not found in animals is photorespiration, which occurs in many plants upon their illumination leading to breakdown of rubisco-1–5-biphosphate and synthesis of glycolic acid in the chloroplast. The glycolic acid produced is oxidized to glyoxalic acid in the peroxisomes with concomitant generation of H₂O₂. Overexpression of the gene encoding glycolate oxidase (see Additional file 1) does suggest enhancement in photorespiration, and catalase (Cat) is probably synthesized at enhanced rate (Fig. 5b, c, see Additional file 1) to protect the plant from oxidative damage by the accumulating H₂O₂. However, so far no relationship between photorespiration, or any of its components, and salt tolerance has been reported.

From the functional characterization of the unigenes, and redundancy of EST in the individual group, it appears that the proteins involved in cell cycle and DNA processing must be playing crucial role in salt adaptation as the redundancy of ESTs in the group was very high, 13.6% (Fig. 7, sub-category C), despite very low contribution by the unigenes (Fig. 6). In the same way, the proteins involved in transcription (sub-category D) and protein synthesis (sub-category E) must also be very important in supporting the plant to go through the salt adaptation processes. This is because the transcription of the genes in these categories greatly increased upon NaCl treatment and contributed individually 7–9% of the ESTs population (Fig. 7) in contrast to each sub-category representing 1.8% of the total unigenes (Fig. 6). The role of the proteins regulating protein activity (sub-category H), however, seems to be very important as the size of the ESTs of this category (Fig. 7) was more than 10 fold the size of the unigenes (Fig. 6) in the group. Besides, the ESTs of the proteins involved in signal transduction (sub-category J) also increased significantly compared to the size of the unigenes in the group. The results thus strongly suggest salt tolerance to be heavily dependent on the expression of the genes contributing to the information pathway (sub-categories C-H) of the plant protein functional catalogue involving protein controlling important cellular functions, such as cell cycle, transcription, protein synthesis, regulation of protein activity, etc. (Fig. 6, 7). Besides, the proteins involved in the developmental processes like cell type differentiation (sub-category Q), biogenesis of cellular components (sub-category P), etc. also appear to play important role in salt tolerance as the EST abundance of the related genes was found to be considerably high after those of the genes of the information pathway. Nonetheless, importance of the other proteins, particularly the unknown ones (sub-category U), cannot be ignored as many biochemical processes determining salt tolerance might be hidden in this pool, although the EST redundancy of the genes encoding these proteins was found to be much less than the size of unigenes in the group.

The present study thus reports for the first time differential expression of genes under salt stress in a natural halophyte. From the number of the unigenes showing overexpression in the plant in response to the salt application, it is highly convincing that the salt tolerance process is highly complex. However, even more puzzling is that a species differs greatly from the other in the genes that are salt regulated, as revealed by a comparative analysis of the present data with that available for rice [19] and tomato [18]. Therefore, it is desirable that more data are generated on the salt inducible/responsive genes of various plant species, particularly of the halophytes in order to identify the key elements involved in the salt tolerance processes, which is not possible with the currently available SSH cDNA libraries related to salt responsive genes. Nevertheless, it is quite convincing that the genes/proteins involved in the flow of information and developmental processes could be of much importance in salt stress response and adaptation of plants to saline environment. The number of genes possibly involved in salt tolerance can be narrowed down further if the SSH cDNA library data on salt inducible genes are available for a sufficiently large number of closely related halophytic species. Once this is achieved, the full length cDNA of the desired genes can be obtained easily and their specific functions in salt tolerance can be investigated further in suitable model systems using the available genetic transformation and gene knockdown/kockout technologies. Functional characterization of the proteins/genes that overexpress under salt stress, but have no known function, will be of particular importance in understanding the complex mechanism associated with salt tolerance. This will eventually enable the biotechnologists to target the right gene(s) for the genetic engineering of crop plants for improved salt tolerance. Nevertheless, the success of such effort will largely depend on the critical number of genes that must be genetically engineered to make a crop plant salt-resistant, besides on the effect of the transformation on the yield quantity and quality of the crop.