RESULTS AND DISCUSSION

Climatic data of the studied areas are shown in Table 1 covering the period from January 1999 to December 1999. The data indicate the intensity of atmospheric drought to which desert plants are subjected. Regarding the climate of the Deltoid portion of Wadi Sudr which exists in south west of Sinai, it was characterized by its great variability. The period from May to August is completely rainless, its monthly values ranged from 2.4 to 6.6 mm during the period of April and December reaching the maximum value of 9.9 mm during January (Table 1).

The irregularity of rainfall is pronounced and this represents a common factor of all arid regions (Strahler, 1951). There are wide variations in air temperature of the south west Sinai area throughout the whole year. Data in Table 1 indicate that the summer months (June, July and August) were the hottest months while those of winter (January, February, November and December) were the coldest ones. The monthly maximum mean temperature reached a maximum of 31.9EC in June. Relative humidity of the Wadi Sudr area recorded its minimum value of 45% in July (1999).

Crynohalophytes

Concerning the climate of the southern area of the Red Sea coast, data in Table 1 indicate that there are wide variations in the rainfall and relative humidity throughout the year. The months March, April and May were the most rainy and humid. According to the climatic records of the Red Sea coastal zone, the mean maximum temperature reached the highest value of 42.1 in August while the mean minimum temperature was recorded in January, 1999 (13.77°C).

Succulent halophytes

The climate of the Red Sea coastal area of Egypt is arid. Many investigators (Zahran and Willis, 1992 and Abd El-Ghani, 1998) have described the climate of the Red Sea coast. Concerning the quaternary ammonium salts of the Crynohalophytes of the Red Sea coastal area, data presented in Table 3 reveal that the osmoprotectant glycinebetaine was accumulated in higher level during summer compared with that recorded in winter in Avicennia marina (310 and 280 n mole g^–1 D.wt.).

Table 1:	Monthly variations of rainfall, maximum and minimum temperatures and relative humidity of Sinai Peninsula and Red Sea regions during the period from January 1999 to December 1999

Atriplex farinosa exhibited a reverse behaviour, where it accumulated the highest amount of glycinebetaine during winter. β-alaninebetaine compound was detected in both Limonium axillare (a) and (b) during winter (38 and 30 n mole g^–1D.wt., respectively). In addition to glycinebetaine both Nitraria retusa (a) and (b) of the Red Sea coast accumulated trigonelline compound during winter (9.26 and 1.413 n mole g^–1D.wt., respectively). Nitraria retusa (a) and (b) of the Sinai Peninsula accumulated very low contents of glycinebetaine during winter (16.428 and 6.667 n mole g^–1D.wt.) compared with those attained by the other plant species. The succulent halophytes species from both Red Sea and Sinai Peninsula areas attained their maximum values during summer which decreased markedly during winter season.

Quaternary ammonium compounds such as glycinebetaine, β-alaninebetaine, trigonel-line act as non toxic (compatible) osomoticum preferentially located in the cytoplasm and chloroplasts of plant cells and also may act as enzyme protectants (Wyn Jones and Storey, 1981). These organic solutes are accumulated in halophytes and in a wide range of glycophytic plant species. The accumulation of quaternary ammonium compounds was highest in the salt tolerant wild species (Baburina and Shevyakova, 1988). Betaines act as osmoregulatory substances, maintaining the osmotic balance with environment (Rhodes and Hanson, 1993).

It is evident that the compatible osmotic solute, glycinebetaine can alter the thermodynamic properties of membranes perhaps by direct interacting with phosphatidylcholine moieties (Rudolph et al., 1986). Moreover, Wyn Jones and Gorham (1983) have discussed the possible roles of glycinebetaine in modulating the flux of Na⁺ from the cytoplasm to the vacuole. They reported that quaternary ammonium salts may specifically modulate the activity of membrane carrier proteins or channels. In addition, betaine compounds stabilize proteins and membranes when present at high concentration and so can be used to raise cytoplasmic osmotic pressure in stressed cells without deleterious effects, since glycinebetaine is predominantly localized in the cytoplasm of leaf cells of halophytic chenopods Suaeda maritima and Atriplex gmelini leaves. Moreover, Rhodes and Hanson (1993) reported that the genes of betaine synthesis are nuclear encoded and are induced by salt/water stress.

β-alaninebetaine is one of the quaternary ammonium salts which was recorded in both Limonium axillare (a) and (b) during winter in response to relatively high total soluble salts (TSS) of the associated soils (Table 2). In plumbaginaceae family, various species accumulated β-alaninebetaine compounds under salt stress which may serve as an osmoprotectant (Hanson et al., 1994). β-alaninebetaine synthesis does not have O₂ requirements, which may be rational for the observation that Limonium species adapted to dry, sandy or rocky soils tend to accumulate glycinebetaine, whereas Limonium species colonizing salt marshes tend to accumulate β-alaninebetaine (Hanson et al., 1991). Interestingly, substituting β-alaninebetaine for glycinebetaine with some species of plumbaginaceae has not narrowed their adaption, as many β-alaninebetaine accumulators are found in dry saline environments as well as the high altitudes. β-alaninebetaine thus appears to be effective over a border ecological spectrum than glycinebetaine (Rhodes and Hanson, 1993). Both of Nitraria retusa (a) and (b) from Sinai Peninsula region accumulated glycinebetaine during winter where trigonelline compounds were not found in these species. Glycinebetaine was induced as a result of high total soluble salts in the soil of both Nitraria retusa (a) and (b) (Table 2). Trigonelline is a pyridine betaine, functions as a cell cycle regulators and accumulates in response to various stresses. It is believed to act as an osmoregulator.

Table 2:	Chemical composition of soil profiles associated with the studied plant species (Hassanein et al., 2003)

Table 3:	Seasonal fluctuations in glycinebetaine, trigonelline, β-alaninebetaine, proline and total soluble protein contents of the studied species under different habitat conditions

Table 4:	Seasonal fluctuations in protein banding patterns of the studied species under different habitat conditions

It is preferentially located in the cytoplasm and plays some other role such as the modification of membrane transport properties. However, it may also be a sensitive indicator of salt stress (Tramontano and Jouve, 1997).

The contents of proline show wide variations among the different species under different habitat conditions (Table 3). It is clear that Nitraria retusa (a) of the Red Sea area attained higher values of proline contents during winter (8.5 μ mole g^–1F.wt.) reaching the maximum value among all the studied plants in Nitraria retusa (a) of the Sinai Peninsula during summer (12.5 μ mole g^–1F.wt.). The succulent halophytes of the two studied areas recorded relatively moderate amounts of proline content ranged between 0.8 to 3 μ mole g^–1F.wt. . Salsola tetrandra (a) and (b) of Sinai Peninsula attained their higher values of proline during winter (2.9 and 1.9 μ mole g^–1F.wt.). These values decreased significantly during summer to 1.5 and 0.8 μ mole g^–1F.wt. Proline is known to be a compatible solute, it accumulates in the cytoplasm without having any detrimental effects on cytosolic enzyme activities and it is the least inhibitory of cell growth among all amino acids (Lutts et al., 1996). Also, proline has been reported to play an important role in osmoregulation as a buffer against osmotic imbalance caused by high vacuolar ion concentration (Serranoo and Gaxiola, 1994). Proline is accumulated by a number of different plants in response to both water stress and salinity (McCue and Hanson, 1990; Delauney and Verma, 1993). Proline accumulation could represent a compensatory mechanism for better survival during the period of drought stress and acted a role as an osmotic regulator (Nikolopoulous and Manetase, 1991). Thus the accumulation of proline in salt stressed plants is considered as one of the major physiological defense mechanisms of these plants against stressed habitats. Limonium axillare (b), Nitraria retusa (a) and (b) of the Red Sea area exhibited significant decreases in proline contents during summer, which followed the effect of relatively high total soluble salts in soil during winter (Table 2).

Table 5:	Seasonal fluctuations in protein banding patterns of the studied species under different habitat conditions

Csonka et al. (1988); Kishor et al. (1995) and Hassanein (2000) suggested that the accumulation of proline in tissues confers some salinity tolerance. Proline may have a significant role to metabolism other than just osmotic adjustment (Hare and Cress, 1997).

The total soluble protein contents in all cyrnohalophytes of the Red Sea area except for Nitraria retusa (b) attained significant increases during summer. Nitraria retusa (b) of the Red Sea area showed the highest value of total soluble protein (4514.9 μg g^–1F.wt.) during winter followed by highly significant decrease to 2034.4 μg g^–1F.wt. during summer. It is of interest to notice that Nitraria retusa (a) of the Red Sea recorded the highest value of total soluble protein among all the studied plants (5241 μg g^–1F.wt.) during summer. From Table 3 it is apparent that both of Nitraria retusa (a) and (b) of the Sinai Peninsula accumulate higher values of total soluble protein during winter (3195.6 and 3445.9 μg g^–1F.wt., respectively). The total soluble protein contents in both Salsola tetrandra (a) and (b) of Sinai Peninsula showed higher values during summer (2198.9 and 1136.5 μg g^–1F.wt., respectively) than those showed in winter (3236.5 and 1709.5 μg g^–1F.wt., respectively). Plants frequently produce a number of unique proteins as a part of their response to environmental stresses (Orcutt and Nilsen, 2000). Salinity also, induced distinct protein changes, which may be either qualitatively or quantitatively (Ramagopal, 1987; Hurkman and Tanaka, 1987).

Several proteins induced by salt stress are also induced by water stress and chilling and the signal transduction pathways may be very similar for all these stresses (Wu et al., 1997). There were variations among the studied plants in protein configuration. It has been shown in the present work that protein pattern of Avicennia marina (Fig. 4 and Table 4) during winter period using one dimensional SDS-PAGE, was characterized by the presence of eleven protein bands which were associated with high total soluble salts in its soil (Table 2).

Table 6:	Seasonal fluctuations in protein banding patterns of the studied species under different habitat conditions

This number of bands were increased during summer to twelve bands with the appearance of new protein bands and the disappearance of others in response to dryness of summer and salt stress. Water stress during the summer may induce the accumulation of stress responsive proteins of molecular weights 112.0, 83.0, 52.0, 34.0, 24.0, 21.0 and 6.5 kD some of these proteins were thought to belong to dehydrins group (25-60 kD) or aquaporins (25.0-30.0 kD) (Arora et al., 2000). Aquaporins are groups of membrane bound proteins that regulate membrane water permeability in cells and may influence water movement among tissues and organs. The permeability of membranes to water depends on the number of aquaporins in the membrane and their effectiveness at transporting water (Maurel, 1997). Aquaporins selectively transport water and do not co-transport any other ions. Therefore, these proteins are important for turgor maintenance and water distribution within and among cells but not so important for ion homeostasis. Dehydrins are predicted to act as stabilizers (chaperon-like) that prevent or reduce the denaturation of other cellular micromolecules under dehydrative conditions (Campbell et al., 1998). Also they have a protective role in the survival of cells under water stress due to their function as ion trap in dehydrating cells sequestering ions as they became concentrated (Close, 1996). Thus the new synthesizing protein bands may be induced for stress adaptation during summer while the disappearance of other bands may be attributed to the degradation of some proteins (Vierling, 1991).

Nine protein bands were detected for Atriplex farinosa (Fig. 4 and Table 4) which ranged from (130.0-5.0 kD) during summer. It is of interest to note that the de novo synthesized protein bands during summer (52.0 and 44.0 kD) have disappeared during winter. These bands may belong to dehydrin group (25-60 kD). These proteins have an important role to plant cell (Close, 1996 and Maurel, 1997). On the other hand, during winter Atriplex farinosa accumulated more protein bands in response to the highest total soluble salts in the associated soil (Table 2), which increased to ten bands ranging from 130.0 to 5.0 kD with the appearance of three new protein bands of molecular weights 99.0, 58.0 and 21.0 kD. In this respect, Godoy et al. (1990) and Vierling and Nguyen (1992) found that responses of plant under salt stress, being characterized by the synthesis of high and low molecular weight proteins.

Protein banding pattern of Limonium axillare (a) attained the same number of protein bands during both seasons. During summer a new protein band (28.0 kD) was induced for the acclimation of this plant to both drought and high total soluble salt stresses during the dry period (Tables 1 and 2). This induced protein may belong to dehydrin group (25-60) or aquaporins (25-30) which have an important role in plant cell (Maurel, 1997). Limonium axillare (b) attained thirteen bands of proteins during summer of molecular weights ranging from (155.0-5.0 kD) and increased to fourteen bands during winter with molecular weights ranging from (186.0-5.0 kD). The new induced protein (186.0 kD) during winter may be one of the adaptive responses of Limonium axillare (b) to salinity conditions.

Acclimation of Nitraria retusa (a) (Fig. 5 and Table 5) to the adverse conditions of drought and salinity in summer has induced seventeen protein bands of molecular weights ranging from 135.0 to 5.0 kD, which decreased to twelve bands during winter. During summer five new protein bands appeared (135.0, 70.0, 44.0, 36.0 and 24.0 kD), which may include dehydrins or aquaporins. These proteins may be important in plant adaptation to the desiccation and ionic effects of saline conditions (Yamaguchi-Shinozaki et al., 1992).

A marked reduction in the number of protein bands was observed in Nitraria retusa (a) grown on coastal regions; 7 km south of Safaga compared with Nitraria retusa (b) grown at 200 m west of the shoreline of Red Sea. Nine bands were recorded during winter of molecular weight ranging from 86.0 to 5.0 kD which increased to eleven one with molecular weights ranged from 122.0 to 5.0 kD during summer. In response to elevation in total soluble salts in soil (Table 2) and dry conditions of summer (Table 1) in Nitraria retusa (b) new proteins were induced with molecular weights of 122.0; 83.0, 28.0 and 16.5 kD. In this concern Zhao and Harris (1992) reported that a large number of genes are induced and accumulated in response to salinity stress, many of which have a known function to help these plants to accommodate stress habitats.

Crynohalophytes from Sinai Peninsula which include Nitraria retusa from two sites (coastal and inland) showed that in summer Nitraria retusa (a) attained thirteen protein bands of molecular weights ranging from 135.0 to 5.0 kD, these bands increased to fourteen bands in winter. The increment in the number of protein bands during winter may be due to the high level of total soluble salts in soil where it reached its highest amount during this period 85760 ppm at (0-20 cm depth) (Table 2). These results indicated that salt stress induced de novo synthesis of stress proteins, suggesting that plant cells were able to monitor different levels of stress intensity and modulate gene expression accordingly these results agreed in part with those of Cherry, (1994). Nitraria retusa (b) showed eleven bands of protein during summer which increased to fourteen ones of molecular weights ranging from 135.0 to 6.5 kD in winter. Three new protein bands were induced during winter of molecular weights 81.0, 62.0 and 28.0 kD, some of these proteins were thought to belong to dehydrins group (25-60 kD) or aquaporins (25.0-30.0 kD). Generally, Nitraria retusa (b) showed a decrease in the number of bands and mostly an increase in their intensities compared with those detected in Nitraria retusa (a). This indicates the diversity of these plants with their different habitats.

Succulent halophytes from Red Sea region as represented by Arthrocnemum glaucum (Fig. 6 and Table 6) attained ten protein bands of molecular weights ranging from 138.0 to 5.0 kD during winter, increased to fifteen ones of molecular weights ranging from 138.0 to 4.0 kD in summer which was associated with relatively high amount of TSS (Table 2). During summer new responsive proteins were induced (130.0, 88.0, 31.0, 25.0, 24.0, 8.0, 6.5 and 4.0 kD), some of these proteins may belong to dehydrins (25-60 kD) or osmotin (25 kD). Close (1996) reported that induction of dehydrins is a general stress response rather than a salt specific response. On the other hand, Osmotin (a type of responsive protein) might play an important role in osmotic adjustment to the cells and adaptive mechanism for salt and drought stresses, either by facilitating rapid accumulation of proline and glycinebetaine localized in the cytoplasm and acts a non toxic osmoticum following osmotic adjustment to occur without perturbing metabolic function (Moons et al., 1995; Orcutt and Nilsen, 2000) or by providing certain metabolic alterations in the cells, which may be helpful in osmotic adjustment (Kononowiez et al., 1993).

Halocnemum strobilaceum (Fig. 6 and Table 6) (Sinai Peninsula) attained fourteen protein bands during summer which increased to sixteen ones ranging between 130.0 to 4.0 kD in winter. During winter some important protein bands were induced like osmotin (25.0kD). Ericson and Alfinito, (1984) reported that upon salinity stress, osmotin can be increased enough to account for 10% of the total protein. In addition to these proteins another kinds of molecular weight proteins 88.0, 35.0, 32.0 and 18.0 kD were observed. These proteins may be induced in response to the high level of TSS (Table 2) during this period which may include aquaporins, dehydrins and other unknown proteins which enable the plant species to accommodate with this stress conditions (Close, 1996).

Species of wide ecological amplitude which include both Salsola tetrandra (a) and (b) (Fig. 6 and Table 6). Salsola tetrandra (a) attained the same number of protein bands during both seasons with no difference in their molecular weights, which ranged from 130.0 to 4.0 kD. On the other hand, Salsola tetrandra (b) showed a higher number of protein bands during winter (ten bands) compared to those attained during summer (seven bands). During summer one of the most adaptive responsive protein was appeared which may be osmotin (25.0 kD) and was associated with the highest amount of total soluble salts in the soil (Table 2). Osmotin may represent induced protein that can be accumulated specifically in response to low water potential and are putatively involved in the maintenance of cellular homeostasis under prolonged stress (Ericson and Alfinito, 1984). Osmotin appeared also to provide osmotic adjustment to the cell by facilitating the accumulation of solutes and providing metabolic alteration in the cells which may be helpful in osmotic adjustment (Singh et al., 1987).

It is of interest to mention that the crynohalophytes Avicennia marina, Atriplex farinosa, Limonium axillare (a) and (b) are characterized by the presence of 4 common salt adaptive proteins of molecular weights 5.0, 21.0-24.0, 28.0-31.0 and 72.0 kD, while Nitraria retusa (a) and (b) grown in Red Sea and Sinai are characterized also by the presence of 4 common salt adaptive proteins of molecular weights 5.0, 11.0, 22.0-24.0 and 28.0-30.0 kD. Succulent halophytes, Arthrocnemum glaucum and Halocnemum strobilaceum share 4 common salt adaptive proteins, their molecular weights are 4.0-5.0, 23.0, 40.0-44.0 and 56.0 kD. In addition, species of wide ecological amplitude which are represented by Salsola tetrandra (a) and (b) grown in Sinai are characterized by the presence of 5 common salt adaptive proteins of molecular weights 4.0-5.0, 21.0-24.0, 29.0-31.0, 35.0 and 56.0 kD.

These results suggest that the production of these stress adaptive proteins may represent a common mechanism which enables halophytes to withstand the harmfull effect of salinity. Some of the salt adaptive proteins may be involved in many functions related to some aspects of protein metabolism such as stabilizing native proteins (Anderson and Guy, 1991), preventing aggregation of denaturated proteins (Sheffield et al., 1990; Ellis and Van der Vies, 1991), refolding stress denaturated proteins (Lee et al., 1995), participating in the folding and assembly of nascent polypeptides into functional forms (Boston et al., 1996), facilitating translocation across membrane (Kang et al., 1990) and targeting non-native and folding incompetent polypeptides for degradation (Chiang et al., 1989). These functions fall under a generalized version of the molecular chaperone concept (Li et al., 1999).

It is evident from the obtained results that creating a favorable water potential gradient is the most challenging factor in the adjustment of the different species to various stress conditions. In this respect, the behavior of halophytes and species of wide ecological amplitude showed different mechanisms in their adjustment to different habitat conditions. The climate of the studied areas is obviously hyper arid. There are wide fluctuations in air temperature, both diurnally and seasonally and scanty rainfall occurs during winter. The variability of rainfall is a feature of both monthly and yearly records.

Quaternary ammonium salts include glycinebetaine, β-alainebetaine and trigonelline which are compatible soluts with the cytoplasm can be accumulated in response to low water availability. This was confirmed by the finding that most of the studied species accumulated high levels of glycinebetaine in response to the high total soluble salts and low moisture availability of the associated soil. β-alaninebetaine, one of the important compatible solutes, was recorded only in Limonium axillare which was associated with the highest value of total soluble salts. Trigonelline, also one of the quaternary ammonium salts, was accumulated in addition to glycinebetaine in Nitraria retusa (Red Sea) in response to the relatively high total soluble salts during winter. Nitraria retusa (Sinai Peninsula) did not accumulate trigonelline which may be explained as a result of the high temperature and climatic aridity in the Red Sea, compared to the climate of Sinai Peninsula. Total protein and proline contents in most of the studied species tended to be accumulated under both salt and drought stresses. Cyrnohalophytes tended to accumulate higher values of proline, but the highest was recorded by Nitraria retusa grown on the coastal region of Delta Wadi Sudr (12.5 μ mol g^–1F.wt) during summer. Such accumulation may be related to more favorable conditions for carrying out metabolic processes. One of the most outstanding features in the adjustment of the studied species under different habitat conditions was the biosynthesis of certain proteins which may play principal roles in the building up of osmotic potential. Regarding protein electrophoresis during both summer and winter in all the studied species (halophytes and species of wide ecological amplitude), some protein bands disappeared and others appeared which may be important in plant adaptation to both salinity and drought stresses. Water stress during summer induced the accumulation of responsive proteins of molecular weights (25-60 kD) which were thought to belong to dehydrins groups. Also, salt stress in most of the studied species was associated with the biosynthesis of a protein with molecular weight 25 kD which might be osmotin. Osmotin play an important role in osmotic adjustment. Some of the induced proteins might be aquaporins which are a group of membrane bound proteins (25-30 kD) that affect water transport through membrane and not ions, their abundance in the tonoplasm makes them important for water and ionic balance between the cytoplasm and the vacuole.

Acknowledgment

The authors wish to express their thanks to Prof. Rhodes, D., Purdue University, West Lafayette, Indiana, USA, for facilitating the analysis of betaine compounds.