1 APS 402 Dissertation Candidate no:000124971 Mechanisms of salt tolerance in halophytes: can crop plants resistance to salinity be improved? Jennifer Seaman High concentrations of sodium are toxic to most plant species, making soil salinity a major abiotic stress in plant productivity world wide. Many crop species, which countless people rely for survival, are negatively affected. Physiological and biochemical research has shown that salt tolerance in halophytes depends on a range of adaptations embracing many aspects of a plants physiology, including; ion compartmentalisation, osmolyte production, germination responses, osmotic adaptation, succulence, selective transport and uptake of ions, enzyme responses, salt excretion and genetic control. The ability of plants cells to maintain low cytosolic sodium concentrations is an essential process associated with the ability of plants to grow in high salt concentrations. New insights into the mechanisms by which plants achieve this have emerged from the identification of genes in Arabidopsis that play a critical role in plant resistance to salt. Unfortunately, there are few investigations which combine studies of growth and other measurements on both biophysical and biochemical plant characteristics. Such joint investigations will be particularly important in the discovery of traits which present the ability to maintain high plant productivity in saline environments. Many questions emerge with rapid advances in the possible genetic transfer of halophyte salt tolerance traits to crop plants, which need to be answered. With several new biological techniques now established, we can begin to plan for rapid progressions in improving crop plant salt resistance in the near future. plants to absorb water [3], causing rapid reductions in growth rate, and induce many metabolic changes [3], similar to those caused by water stress. If tolerance cannot be improved then vast amounts of soils may be left uncultivated. Halophytes are plants growing on or surviving in saline conditions, such as marine estuaries and salt marshes (Fig.1). They respond to salt stress at three different levels; cellular, tissue and the whole plant level [3]. Therefore, in order to successfully understand salt tolerance in plants, the mechanisms at each level must be studied individually. Both ecologists and plant physiologists have long been interested in the effect of salinity on plants. The most recent reviews include those by; Epstein; Flowers & Yeo; Flowers, Hagibagheri & Clipson; Goodin ;Greenway & Munns; Waisel; Wyn Jones et al., and Yeo [3, 4-12]. However, little extensive research has been carried out since to further our understanding of how such plants respond to saline conditions. To achieve salt tolerance three interconnected aspects of plant activity are important. Damage must be prevented, homeostatic conditions must be re-established and growth must resume (Fig.2). Seven percent of the land’s surface and five percent of cultivated lands are affected by salinity [1], with salt stress being one of the most serious environmental factors limiting the productivity of crop plants [2]. Therefore, extensive research into plant salt tolerance has been carried out, with the aim of improving the resistance of crop plants. With a large majority of the world’s population relying on crops such as barley, maize and rice to survive, crop salt tolerance is globally important [2]. Fig 1. The halophyte Spartina anglica. [4] Despite advances in increasing plant productivity and resistance to a number of pests and diseases, improving salt tolerance in crop plants remains elusive, mainly because salinity simultaneously affects several aspects of plant physiology. Saline conditions reduce the ability of 2 APS 402 Dissertation Candidate no:000124971 Growth and survival of vascular plants at high salinity depends on adaptation to both low water potentials and high sodium concentrations, with high salinity in the external solution of plant cells producing a variety of negative consequences. Salt stress causes ionic imbalance [14], with excess sodium and chloride ions having a deleterious effect on many cellular systems [15], therefore, plant survival and growth depends on adaptations to re-establish homeostasis. High salinity also inflicts hyper osmotic shock on plants, as chemical activity of water is decreased, causing a loss of cell turgor. Salt induced reduction in chloroplast stromal volume and generation of reactive oxygen species (ROS) also plays an important role in decreasing plant photosynthetic capacity [16], and therefore growth. Due to such factors, many halophytes demonstrate reduced growth at high salinity (Fig.3). Fig. 2. The three aspects of salt tolerance in plants (homeostasis, detoxification and growth control) and the pathways that interconnect them; homeostasis is broken down into ionic and osmotic homeostasis. The SOS pathway mediates ionic homeostasis and Na + tolerance. The two primary stresses, ionic and osmotic stresses, cause damage or secondary stresses such as oxidation. Lea-type stress proteins such as RD29A are proposed to function in the detoxification or alleviation of damages. CBF/DREB transcription factors mediate some of the stress protein gene expression in response to secondary stresses caused by high salt concentrations, cold, drought or abscisic acid (ABA). The ionic homeostasis, osmotic homeostasis and detoxification pathways are proposed to feed actively into cell division and expansion regulation to control plant growth. [13] The complexity of higher plants causes problems in experimentation and interpretation of results, making it difficult to decipher the exact effect that salt exerts on plants. Problems are associated with measurements of ion and water relations, as well as growth rates in plants subjected to salt stress [18]. Most growth data are based on single harvests, causing problems as vascular plants often change in growth rate with time. Due to the massive amounts of minerals in plant leaves interpretation of growth rates in terms of organic dry weight, in plants grown at high salt concentration, is undermined [19]. Further problems are caused as certain halophytes have a nutritional requirement for sodium. Interpretation of the cause for increased growth in such species, with increasing salt concentration, is hampered in experiments in which the control nutrient solutions contain only trace amounts of chloride and sodium [18]. Fig.3. Scheme showing possible causes of reduced growth of vascular halophytes at high external NaCl. [17] Given all the factors determining the pleiotropic deleterious effects of salinity on plants, it is not surprising that adaptation to salinity involves the modification of a large number of parameters. At present numerous mechanisms of salt tolerance in halophytes are proposed and will be discussed in this review, including ion compartmentalisation, osmolyte production, germination responses, osmotic adaptation, succulence, selective transport and uptake of ions, enzyme responses, salt excretion and genetic control. 3 APS 402 Dissertation Candidate no:000124971 Cellular adaptations of halophytes to high salinity Osmotic adaptation – water relations and osmolyte production Salt negatively affects the water flow towards plant roots, owing to a decrease in water permeability of the soil, and reduces water conductivity of plant roots. As a result, cell membrane permeability drops and influx of water to the plant is reduced [9]. A metabolic response to salt stress is the synthesis of compatible osmolytes. These mediate osmotic adjustment and therefore protect sub-cellular structures and reduce oxidative damage caused by free radicals, produced in response to high salinity [20, 21]. Osmolyte compounds include sugars, polyols, amino acids and tertiary and quarternary ammonium, and sulphonium compounds [22]. Glycine betaine (GB), a quarternary ammonium compound, is a stabilizing osmolyte, helping to preserve macromolecules under dehydration stresses, giving rise to its name as an ‘osmoprotectant’ [23]. In many species of halophytic Poaceae and Chenopodiceae GB is seen to accumulate, but does not do so in most crop species [6]. Therefore, considerable work has been carried out to try to engineer GB production, which is synthesised from betaine aldehyde dehydrogenase (BADH), in species that do not produce it naturally. For example, in sugar beet, BADH activity increased 2-4- fold in its roots and leaves as sodium chloride concentration was raised from 0 to 500 mM [24]. Oxidation of choline to BADH (the predominant biosynthetic route in all betaine producers) [22], is catalysed by a soluble ferrodoxin-dependent monoxygenase (CMO) in the chloroplasts of higher plants, or by a poorly characterized membrane associated choline dehydrogenase [25]. Transgenic tobacco plants harbouring the E.coli beta gene, encoding a choline dehydrogenase, were more tolerant to salt conditions than wild type plants [26], demonstrating its role in salt tolerance. Some of the Australian halophytic Melanleuca species are salt tolerant, which has been attributed to their ability to accumulate large quantities of osmoprotectants known as proline analogues [27]. These have been used in seed treatment and foliar application to increase the salt resistance of economic crops. Melanleuca bracteata, which accumulates the proline analogue 4-hydroxy-N- methyl proline (MHP), could potentially be harvested for commercial use, playing a positive role in the control of dry land salinity, with an average yield of 393kg/ha of MHP, correlating to a gross economic return of AU$14505/ha [27]. However, there has been little gain for plants in the field from attempts at engineering GB and MHP probably due to their regulation, compartmentalisation, and production outlay [28]. Recent elucidation of a class of membrane proteins termed aquaporins, hints at the existence of some possibility for intracellular compartmentalisation of water [29]. In halophytes these pore-forming proteins conduct water molecules across membranes, implicating that gating of water channels could have an impact on intercompartmental movement of water [29]. In terms of salt tolerance, such aquaporins could be a mechanism for maintaining osmotic homeostasis and turgor in the cells of salt stressed plants. Protection of cell wall integrity Cell wall properties, such as permeability, are involved in the maintenance of cell growth during salt stress [30] and could be crucial to salt tolerance. Overexpression of TPX2, a cell wall peroxidase in tomato, significantly increased germination rate under salt stress [31]. The higher capacity of transgenic seeds to retain water could result in higher germination rates in conditions where water supply is limited [31], such as in saline conditions. Salinity generates an increase in reactive oxygen species (ROS), which have deleterious effects on cell metabolism [32, 33]. The potential role of superoxide dismutase (SOD), which eliminates ROS, has been investigated [34]. At high salinity transgenic plants had a total SOD activity about 1.5 times that of the control plants [34], implicating it may play role in salt tolerance. The role of photorespiration in stress conditions is still controversial, but may function as a dissipation mechanism for excess light energy or reducing power [35]. The rate-limiting step in photorespiration is the reassimilation of ammonia catalysed by chloroplastic glutamine synthetase (GS) [36]. When rice plants were transformed with the GS2 gene [37], they accumulated about 1.5 times GS2 than control plants, had increased photorespiration capacity and enhanced salt tolerance [37]. Additional research is needed to confirm the extent of what such mechanisms play in salt tolerance. Enzyme responses A number of enzymes isolated from halophytes are sensitive to sodium chloride (NaCl) when assayed in vitro, at lower concentrations of NaCl than were present in their tissues as a whole [38]. Translocation systems, as well as individual enzymes were affected, being completely inhibited in 300 mol m-3 monovalent cation [39], showing in vitro a narrow concentration range of ions essential for mRNA synthesis. Ionic shifts as small as 10mol m-3 potassium or 1 mol m-3 magnesium above or below the optimum causes a decrease in the absolute rate of translation in vitro [40]. In the highly vacuolated tissues of Suaeda maritima concentrations of 0-250 mM Cl- and Na+ have been reported in chloroplasts and 280mM in the cytoplasm [17]. These concentrations are high considering in vitro the activity of many enzymes, including those involved in protein synthesis, are inhibited at 200-400 mM NaCl [17] This suggests that a higher salt tolerance of metabolism in vivo rather than in vitro may occur due to increase in substrate concentration, and increase or decrease in levels of enzymes involved in intermediate metabolism. The ability of halophytes to grow in highly saline conditions 4 APS 402 Dissertation Candidate no:000124971 probably involves such subtle changes, as there is little evidence that there is any inherent difference in salt tolerance between enzymes isolated from halophytes and non-halophytes. Ion compartmentalisation and selective transport and uptake of ions at the plasma membrane Internal salt concentrations can reach levels three times that of the external medium [17]. In the absence of salt regulating mechanisms, this uptake would result in the shoot essentially becoming a pillar of salt! Therefore, the ability of plant cells to maintain low cytosolic sodium concentrations is an essential process for halophytes [41]. NaCl is excluded from the phloem, therefore not reaching plant flowers and seeds internally. However, leaves being fed by the transpiration stream, receive large quantities of sodium, which must be regulated [42]. Plant cells respond to salt stress by increasing sodium efflux at the plasma cell membrane and sodium accumulation in the vacuole. For such a reason, the proteins, and ultimately genes, involved in these processes (discussed later in this review) can be considered as salt tolerance determinants [41]. Recent studies have identified pathways for sodium entry into cells [43] and the cloning of Na+/H+ antiporters [44] have demonstrated the role of intracellular sodium compartmentalisation in plant salt tolerance. Such compartmentalisation of sodium and chloride in leaf vacuoles can only be attained if (1) sodium and chloride ions are actively transported into the vacuole and if (2) tonoplast permeability to these ions is kept low enough to sustain the ion concentration gradients at an energy cost that can be prolonged for many months [45]. Tonoplast conductance of higher plants can become very large, owing to the opening of unselective, slow vacuolar (SV) ion channels, as demonstrated by many patch clamp investigations [46]. SV channels conduct both chloride and sodium and would therefore tend to dissipate a NaCl concentration gradient across the tonoplast, causing plant death, if conductance for sodium and or chloride rises above the capability of active transport to prevent such a leakage [45]. Halophyte tonoplast channels must therefore be modified either to be increasingly discriminating against sodium and chloride, or to have channels closed for the greater part of time, or to have a decreased number of channels per cell. It has been found that in halophytes in vitro ATP-dependent proton pumping capacity was comparable with that of non-halophytes [45], whether grown in saline conditions or not, and that the most common ion channel was the SV type. Selectivity for sodium and Chloride and potassium was just as small as in glycophytes, despite its importance to minimise leakage out of vacuoles [47]. It is concluded that leakage is reduced by a very low open probability in saline conditions, therefore needing no specific adaptation in SV channel trait in halophytes. The gating frequency of such channels is very low (Fig.4). Further study may reveal why higher plants tonoplasts contain such great numbers of SV type channels. As an increase in opening frequency in halophytes is fatal, it can only be concluded that the opening frequency of these channels in vivo is equally as low [46]. Fig.4. Voltage dependence of the open probability in Suaeda maritima. Gating of the SV channel is strongly voltage- dependent and open probability increases at negative tonoplast potentials. [45] The transport of ions across the plasma membrane and tonoplast requires energy, which is provided by vacuolar and plasma membrane ATPase [48]. Sodium ions are exchanged for hydrogen ions across a membrane as membrane Na+/H+ antiporters take advantage of the proton gradient formed by these pumps [49] (Fig.5). Salt stress was shown to increase Na+/H+ activity in glycophytes and halophytes [50, 51]. The activation of such antiporters is likely to be operating to reduce sodium toxicity in salt tolerant plants under saline conditions. Figure.5. Proton pumps and antiporters of plasma membrane and tonoplast. The primary active P-ATPase energizes the plasma membrane for secondary active transport of Na + by Na + /H + antiport to the cell wall, and the primary active V- A TPase and V-PPase energize the tonoplast for secondary active transport of Na + by Na + /H + to the vacuole. [52] 5 APS 402 Dissertation Candidate no:000124971 A salt-sensitive rice cultivar (Oryza sativa cv. Kinuhikari) has attempted to be engineered that expresses the vacuolar-type Na+/H+ antiporter gene from the halophytic plant Atriplex gmelini (Ag NHX1) [44]. In the transgenic rice plants, the activity of the vacuolar Na+/H+ antiporter was eight times that of wild type rice [44]. Transgenic plants over expressing AgNHX1 could survive under conditions of 300mM NaCl for three days, whereas to wild type plants, this treatment was fatal [44], indicating that over expression of the Na+/H+ antiporter significantly enhances transgenic rice salt tolerance. Extending such research could improve crop plant salt tolerance in the near future if successful genetic transfer can occur on a large scale. Salt tolerance requires not only the adaptation to sodium toxicity, but also the acquisition of potassium (an essential nutrient) [52] whose uptake is affected by high external sodium concentration, due to the chemical similarity of the two ions. Therefore, potassium transport systems involving good selectivity of potassium over sodium can also be considered an important salt tolerance determinant [52]. Salt stress, along with water and cold stresses [53], increases biosynthesis and accumulation of abscisic acid (ABA) in higher plants. For example, ABA content increased only in the leaves of a salt tolerant rice cultivar versus the sensible cultivar. The increased ABA content was accompanied by an improvement in K+/Na+ ratio [54], leading to increased salt tolerance. The factor responsible for initiating such an increase in ABA biosynthesis remains elusive, requiring further research. Inconsistencies exist in some studies aiming to identify mechanisms for ion regulation in halophytes, which are possibly due to differences in cultivars, experimental conditions, and salt level used and age. It is obvious that further study is required to clarify exactly the mechanisms for compartmentalisation in halophytes, before they can begin to be applied to crop plants. Genetic control: Pass the salt please: Arabidopsis as a model system With the advent of molecular biology, a common approach used to define salt tolerance mechanisms in plants has been to identify cellular processes and genes whose activity or expression is affected by salt stress [14,55-58], which has led to a better understanding of the complexity of salt tolerance in higher plants. Recently, the glycophyte, Arabidopsis, has emerged as an excellent model system to study plant salt tolerance [43]. Application of this model has yielded a regulatory pathway for ionic homeostasis under salt tolerance [43]. Salt overly sensitive (SOS) genes were cloned, with mutations of these genes rendering Arabidopsis more sensitive to salt stress. SOS3 interacts physically with SOS2 [59]. SOS1 is a downstream target of the SOS3- SOS2 kinase complex and is a plasma membrane Na+-H+ antiporter that exports sodium from the cell (Fig.6). The upregulation of SOS1 expression by salt stresses in wild type Arabidopsis plants is reduced by SOS3 or SOS2 mutations [60]. SOS1 over expression is a promising new approach to engineer salt tolerance in crop species. Once the region in SOS1 mRNA that determines salt stress regulated stabilisation is identified, it can be used to engineer more precise regulation for other genes used to confer salt resistance [60]. Evidence suggests that a protein kinase complex of SOS3 and SOS2 is activated by a salt stress elicited calcium signal [61].This protein kinase complex then phosphorylates and activates various ion transporters, such as the plasma membrane Na+/H+ antiporter SOS1 (Fig.6.) [61]. Fig. 6. Model for signal transduction in Arabidopsis under salt-stress conditions. Pathways for Na + influx, leading to Na + toxicity, include non-selective cation channels and HKT1. SOS3 is a Ca 2+ sensor homolog that activates SOS2, a protein kinase that, in turn, phosphorylates and activates the plasma membrane Na + /H + antiporter SOS1. SOS1 mRNA is stabilized and accumulates under salt stress conditions. Over-expression of the vacuolar Na + /H + antiporter NHX1 also confers salt tolerance in plants and expression of NHX1 is up regulated in SOS mutants, probably through indirect effects on ion homeostasis. [64] Two allelic recessive mutations in Arabidopsis, sas2-1 and sas2-2, which induce sodium over accumulation in shoots, have been defined [62]. The sas locus corresponds to the HKT1 gene, which is a sodium influx transporter in Arabidopsis. Expression in Xenopus oocytes showed that sas2-1 mutation did not affect the selectivity of this transporter, but greatly reduced transport activity [62]. In Arabidopsis HKT1 expression is restricted to the phloem tissue in all organs, with sas2-1 mutation strongly reducing sodium concentration in the phloem sap, leading to sodium over accumulation in every aerial organ, except the stem, but no under accumulation in roots. Sas2 plants displayed increased sensitivity to NaCl, with reduced growth and whole plant death under moderate salinity [62]. These results suggest that HKT1 is involved in the recirculation of sodium from the shoots to the roots, and is probably achieved by mediating sodium loading into the phloem sap in shoots and unloading in the roots [62]. For such a reason, HKT1 could play a vital role in plant salt tolerance. 6 APS 402 Dissertation Candidate no:000124971 Succulent leaves also have more mitochondria. For example, a bean plant treated with Nacl (0.2M) had approximately six times as many mitochondria than control plants [71]. The mitochondria of salt affected plants were also larger; evidencing that extra energy is needed in these plants for salt compartmentalisation and excretion [71]. Salt stress inhibits cell growth; a possible cause being lowered photosynthesis as stomata close to reduce water loss, limiting carbon dioxide uptake [77] (discussed later in the review). Cell expansion and division may also be affected directly by salt stress. An important link between stress and cell division was revealed by induction of the ICK1 gene (a cyclin-dependent protein –kinase inhibitor) in Arabidopsis by abscisic acid (ABA) [63]. ICK1 may inhibit cell division by reducing the activities of cyclin- dependent protein kinases that drive the cell cycle. Under salt stress ABA accumulation may induce ICK1 activity [63]. The constitutive expression of several stress-related genes including CBF1, DREB1A and ATHB7 has been shown to cause slow growth in transgenic plants [64-66]. These genes respond to cold and drought stress and are not usually expressed under normal growth conditions, implicating their role in plant salt tolerance. The halophytic plant species Thellungiella halophila, which can survive at seawater level salinity, is closely related to Arabidopsis (DNA sequence of 90% or more similarity [13]). They share a similar morphology and life history. T.halophila plants are transformed by the proven ‘floral dripping’ method and it is possible to generate hundreds of thousands of T-DNA insertion lines of clone mutations affecting salt tolerance [67]. Through systematic genetic analysis T.halophila has the potential to enable the determination of salt tolerance determinants and pathways operating in halophytes [67]. Fig.7 Cross sections of Suaeda monoica leaves taken from plants grown under saline (S) and non-saline (ns) conditions. [9] However, despite the promise of molecular studies with Arabidopsis, no successful transgenic crops have so far been produced. There are two possible reasons for this. Firstly, the reductions in yields due to salinisation may not to date have become a problem of sufficient importance for ecophysiologists to give high precedence to the creation of new salt tolerant cultivars. Secondly, the limited success of engineering programmes may relate to the polygenic nature of the salt tolerance trait [68]. However, with increasing soil salinity world wide, it will become ever more important to allocate time and money into determining salt tolerance traits in halophytes for genetic transfer to crops. It is debatable as to whether succulence is simply a response to salinity or whether such characteristics are responses to adaptive value [9]. As halophytes become succulent in response to increasing salinity, and such influential changes seem to be an integral part of halophytic development, it is tempting to presume that such modifications are of adaptive value [9]. It is important to point out, however, that some halophytes do not become succulent at all in response to increasing salinity. Only further study will determine whether succulence is in fact a response or an adaptation to salinity in halophytes. Salt excretion Tissue adaptations of halophytes to high salinity Succulence One of the most noticeable features of halophytes is the correlation between uptake of alkali ions and whole plant succulence [9], with the aim of balancing out ion toxicity created in saline conditions by increasing the total plant water content. Succulence is seen as an increase in cell size, decrease in extension of growth, decrease in surface area per tissue volume, and high water content per unit of surface area [71-73] (Fig.7). Succulent plants have thick leaves, mainly owing to an increase in the size of their mesophyll cells. Such leaves also have smaller intracellular spaces (Fig.7.). Halophytes utilise salts in osmotic adjustment to the low water potentials of their environments. They must accumulate sufficient ions in their leaves for this purpose, whilst avoiding the toxic effect of those ions [8]. In some species, growth and ion accumulation are balanced [72] while in others excess ions are secreted via salt glands [73]. Mineral content of shoots is best regulated by the secretion of ions through specialised salt glands, such as in Spartina species. However, salts are also released through the cuticle or in guttation fluid; they are retransported back to the roots and soil via the phloem, or become concentrated in salt hairs [74]. Salt glands are located on or depressed into the epidermis and are found in almost 7 APS 402 Dissertation Candidate no:000124971 every aerial part of the plant, but tend to be concentrated on the leaves (Fig.8). Salt glands lack a central vacuole and have a high number of mitochondria and other organelles, suggesting that they act primarily not as storage cells, but as transit cells [9]. Considerable amounts of fluid can be excreted by the glands, for example, in Limonium latifolum, leaf disks under certain conditions secreted fluid up to half their weight in a 24-hour period [9]. Ion selectivity also occurs in salt glands. For example, in Sporobolus spicatus, sodium and chloride were the dominant ions in the soil, and together comprised 93% of the dry weight of secreted salt [75]. The molar ratio of potassium: sodium in the plant leaves was more than ten- fold that in the interstitial soil solution and thirteen times that in the secreted salts, reflecting the high selectivity of the secretion mechanism for sodium [75]. Discard of salt saturated organs also removes large quantities of salt from some halophytes. Allenrolfea, Halocnemun and Salicornia discard parts of their fleshy cortex and leaves in order to remove excess salt [76]. However, not all halophytes have salt glands, neither do they all discard salt saturated tissue, demonstrating that individual halophytes utilise different salt tolerance traits in different situations. Due to the existence of such a wide range of salt tolerance traits in halophytes, being applied differently depending on the individual plant and its surrounding environment, it makes the job of determining the exact mechanisms regulating such traits complicated. Stomatal responses Although there are few data available, it is possible to identify two stomatal adaptations to salinity (1) the guard cells can utilise sodium instead of potassium to achieve their normal regulation of turgor (2) the guard cells continue to use potassium and are able to limit their intake of sodium [77]. This mechanism may be very important in glandless halophytes, lacking secretion mechanisms, and it may therefore be of particular interest as a potential contributor to the development of salt tolerance in crops. Fig.8. Segments of a leaf cross section of Avicennia marina, showing sunken glands in the upper epidermis (a) and elevated glands on the lower one (b). [9] Stomatal movements are brought about by changes in the turgor pressure of the guard cells, which, in the vast majority of cases examined, results in fluxes of potassium across the plasma membrane and tonoplast [77]. In an experiment, stomata of the glycophyte Commenalina communis opened as readily in the presence of NaCl as they did in KCl [78] (Fig.9). However, thereafter the stomata showed greatly reduced closing responses to darkness, CO2 and abscisic acid (Fig.10). If this were to occur in saline conditions, the control of gas exchange could be seriously inhibited and even fatal. Fig.9. The effect of changes in concentration of NaCl and KCl on stomatal opening in detached epidermis of Commelina communis, in light and in the absence of CO 2 . [77] Sodium can substitute for potassium in the stomatal mechanism [79]. In the guard cells of salt grown Suaeda maritima, sodium was the major cation, with lower concentrations in the guard cells of closed stomata. In contrast to S.maritima, in Aster tripolium, there were substantial amounts of potassium in the guard cells of salt- grown plants [79]. Potassium remained the dominant cation in the cell, suggesting that the guard cells must have mechanism for restricting the entry sodium, therefore controlling transpiration, which is important as A.tripolium lack salt glands. The inhibition of stomatal opening by sodium provides a ‘top-down’ regulatory mechanism for the control of salt burden in the shoot, when a plants capacity to compartmentalise it is exceeded [80]. In glycophytes, stomatal function is damaged by sodium ions, and this disruption can be seen as a mechanism of their lack of survival in saline conditions [77]. 8 APS 402 Dissertation Candidate no:000124971 It is concluded that guard cells would became irreversibly damaged if they were allowed to accumulate excessive amounts of sodium ions [81]. The introduction of sodium ions into guard cells blocks the outward potassium channels, stopping stomatal closure, essentially locking stomata open. Evidence exists demonstrating that the stomata of non-halophytes may be disabled if sodium enters the cytoplasm of guard cells, suggesting that halophytes must as, one of their adaptive features, developed different stomatal ionic properties from those of glycophytes. Fig.10. The effects of light, CO 2 and ABA (10-5 mol m-3) on stomatal opening on detached epidermis of Commelina communis in the presence of KCL or NaCl (50 mol m-3). [77] Stomatal responses playing a role in salt tolerance are admittedly speculative, but are based on an expanding knowledge. In the future, it is important to monitor more closely such sub-cellular responses, as well as whole plant level responses. Whole plant level adaptations of halophytes to high salinity Germination Reponses Halophyte germination is affected in two ways when seeds are exposed to saline conditions. Firstly, the high osmotic potential of the medium prevents the embryo from taking up water, and secondly, the toxic effect of some ions leads to embryo poisoning [82]. Laboratory investigations indicate that seeds of most halophytic species reach maximum germination in distilled water [83]. Seed germination in saline environments usually occurs during the spring or in a season with high precipitation, when soil salinity levels are usually reduced [84]. In general, both halophytes [85] and glycophytes [86] respond in a similar manner to increased salinity stress; with both a reduction in the total number of seeds germinating and a delay in the initiation of the germination process. Seeds of many species, including Atriplex halimus and Crithmum maitiumum remain dormant at low water potentials [83]. These seeds do not lose their viability and will germinate when returned to a distilled water treatment, indicating that no permanent specific ion toxicity is produced and that the primary influence of excess salts may be osmotic [83]. This recovery response is common among halophytes implying an ecological significance within highly saline environments, reflecting a physiological response that is strongly selected for during the evolution of halophytic species. An important effect of the release of dormancy with the alleviation of salt stress is that it determines the salinity level at the period of seedling development, one of the most sensitive periods in the life cycle of halophytes, to ensure survival of the developing seedlings [83]. NaCl priming in melon seedlings grown in saline conditions increases their salt tolerance [87]. Total emergence and dry weight were higher in melon seedlings derived from primed seedlings and they emerged earlier then non-primed seeds. NaCl priming enhanced total sugar and proline accumulation and prevented toxic and nutrient deficiency effects of salinity as less sodium, but more potassium and calcium was accumulated in melon seedlings [87]. Na: Ca balances were significantly lower in primed seeds under identical salinity levels, suggesting that NaCl priming in melon seeds increases salt tolerance by promoting potassium and calcium accumulation and inducing osmoregulation by the accumulation of organic solutes [87]. 9 APS 402 Dissertation Candidate no:000124971 Reports of salt tolerance at germination level in halophytes have failed to take into account differences in salt tolerance during early seedling development. An investigation showed that germinating seeds in both a halophyte and a glycophyte were salt tolerant up to the erection of the hypocotyls (the stalks of small seedlings) [88]. Beyond that stage the halophyte remained salt tolerant, but the glycophyte became salt insensitive. It seems that although capable of germinating on salt marsh to a certain extent, glycophyte seedlings are not able to compete with halophytic species [89]. Nevertheless, germination of halophyte seeds is inhibited or severely reduced at salinity levels above 250mM NaCl [88]. Interactions between temperature, hormones, salinity and seed germination optima may also exist in halophytes [84], which should be further investigated. The bottom line is that NaCl inhibits germination of many plants, including some halophytes. However, overall halophytes are better tailored to germinate under saline conditions. If the definite mechanisms of salt tolerance at the germination stage can be defined, they will be extremely valuable in improving crop plant resistance to salinity. Conclusion The conclusion was drawn long ago that halophytes differed from glycophytes quantitatively rather than qualitatively [9]. With the exception of salt excreting glands there is no clear distinctive attribute of salt tolerant plants. However, halophyte compartmentalisation is clearly superior, water use efficiency is increased and ion selectivity is probably improved. Some key metabolic effects may also be more tolerant. Controversial data exists regarding the question of whether halophytes require saline conditions for their existence or merely tolerate them [2, 12, 90]. It seems that the biochemical mechanisms leading to salt tolerance in halophytes are regulated in such a way that allow them a competitive advantage to salt stress over other plants, such as glycophytes [57,58]. The majority of halophytes grow well under freshwater conditions, with many showing optimal growth, but even so, glycophyte growth rates are likely to be higher. Halophytes are thus at a competitive disadvantage great enough to eliminate them from favourable fresh water sites [91, 92]. Mechanisms in halophytes, allowing them to survive saline conditions include; regulation of ion concentrations, osmotic adaptation, enzyme adaptation, production of osmolytes, stomatal adaptation, germination responses, ion compartmentalisation and some form of genetic control. However, individual plants will vary in the traits that they possess and to the extent of which they are utilised. Regulation of sodium, chloride, and potassium uptake is almost certainly vital in the adaptation of plants to saline soils [17]. At high rates of transpiration, the xylem of all species contains much lower chloride and sodium concentrations than the external saline environments. Amongst halophytic species low water potentials are controlled largely by ion accumulation; being sequestered in the vacuoles and stabilised in the cytoplasm by organic solutes. The compartmentalisation of ions and organic solutes necessary for salt tolerance occurs at cellular, tissue and organ levels of organisation, with recent work demonstrating that genetic control may play a key role. As an outcome, salt tolerance is rather a function of the whole plant than of isolated cells or tissues [93]. Most research into the effects of salinity on plants has investigated changes occurring in leaves. However, it is the roots which are in direct contact with the saline solution. Although there are opportunities to control salt entering leaves at various points along the transpiration stream [94], the roots must perform a crucial function in the management of input and throughput. It therefore needs to be investigated as to how the activities of the roots and the shoots are incorporated. While in laboratory conditions, halophytes can produce tissue water potentials lower than those in the rooting solution. However, in the field, salinities are variable [12]. How rapidly do plants respond to such changes? Uptake of ions and synthesis of osmolytes will not be instantaneous. The outcome of such questions could be crucial for plant salt tolerance. An important question that needs to be answered is whether glycophytes are capable of retaining halophytic traits without a loss in their productivity? Transgenic approaches for increasing plant salt tolerance are possible, and so far results obtained with many genes are promising. However, since many plants differ in response to stress it should be noted that this is merely the beginning. Transformation of agronomically important crops and the identification of uncovered tolerance traits and stress inducible promoters must be further explored to produce successful salt tolerant crops, as despite to promise from molecular biology, the listing of new cultivars of crop species possessing functional degrees of salt tolerance is diminutive. In the near future, however, with the degradation of irrigation systems, increasing the salt resistance of many crops will become essential [68]. The increasing use of poor quality water, additions of waste salts to our environment, as well as increasing contamination of underground water sources, is leading to a steady soil contamination [2]. Plants capable of existing in such conditions are predominantly halophytic, yet even after years of research, still relatively little are known about their survival mechanisms. 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