CC BY
137
Journal of Stress Physiology & Biochemistry, Vol. 9 No. 3 2013, pp. 5-22 ISSN 1997-0838 Original Text Copyright © 2013 by Aldesuquy, Abbas, Abo-Hamed, Elhakem
ORIGINAL ARTICLE
Does Glycine Betaine and Salicylic Acid Ameliorate the Negative Effect of Drought on Wheat by Regulating Osmotic Adjustment through Solutes Accumulation?
Heshmat S. Aldesuquy1, Mohamed A. Abbas1, Samy A. Abo-Hamed1 and Abeer H. Elhakem2
1 Department of Botany, Faculty of Science, Mansoura University, Egypt.
2 Department of Biology, Faculty of Science, Salman Bin Abule Aziz University, KSA.
*E-Mail: [email protected]
Received December 1, 2012
A pot experiment was conducted to evaluate the beneficial effect of foliar application of glycine betaine (10mM), grain presoaking in salicylic acid (0.05 M) and their interaction on drought tolerance of two wheat (Triticum aestivum L.) cultivars (sensitive, Sakha 94 and resistant, Sakha 93). Osmotic pressure, some osmolytes concentration and grain yield were determined. Water stress caused an increase in osmotic pressure, proline, total soluble nitrogen, total soluble sugars, organic acids, ions (Na+, K+, Ca+2, Mg+2 and Cl") content as well as Na+/K+ ratio in cell sap flag leaves of both wheat cultivars. The resistant variety had higher values of osmotic pressure, proline, organic acids and ions content than the sensitive one. On the other hand, water stress induced marked decrease (P<0.05) in grain yield. The applied chemicals mitigated the effect of water stress on the used wheat cultivars. The effect was more pronounced with glycine betaine + salicylic acid treatment. The applied chemicals increased the osmotic pressure, the osmolytes concentrations as well as the grain yield. Furthermore, the osmotic pressure of flag leaf sap appeared to depend on proline, TSN, TSS, organic acids and the ions content. The economic yield (grain yield) was positively correlated with proline, keto-acids and osmotic pressure but negatively correlated with TSN, TSS and citric acid.
Key words: (Triticum aestivum L.)/drought/glycine betaine/ osmolytes/salicylic acid/yield
ORIGINAL ARTICLE
Does Glycine Betaine and Salicylic Acid Ameliorate the Negative Effect of Drought on Wheat by Regulating Osmotic Adjustment through Solutes Accumulation?
Heshmat S. Aldesuquy1, Mohamed A. Abbas1, Samy A. Abo-Hamed1 and Abeer H. Elhakem2
1 Department of Botany, Faculty of Science, Mansoura University, Egypt.
2 Department of Biology, Faculty of Science, Salman Bin Abule Aziz University, KSA. *E-Mail: [email protected]
Received December 1, 2012
A pot experiment was conducted to evaluate the beneficial effect of foliar application of glycine betaine (10mM), grain presoaking in salicylic acid (0.05 M) and their interaction on drought tolerance of two wheat (Triticum aestivum L.) cultivars (sensitive, Sakha 94 and resistant, Sakha 93). Osmotic pressure, some osmolytes concentration and grain yield were determined. Water stress caused an increase in osmotic pressure, proline, total soluble nitrogen, total soluble sugars, organic acids, ions (Na+, K+, Ca+2, Mg+2 and Cl") content as well as Na+/K+ ratio in cell sap flag leaves of both wheat cultivars. The resistant variety had higher values of osmotic pressure, proline, organic acids and ions content than the sensitive one. On the other hand, water stress induced marked decrease (P<0.05) in grain yield. The applied chemicals mitigated the effect of water stress on the used wheat cultivars. The effect was more pronounced with glycine betaine + salicylic acid treatment. The applied chemicals increased the osmotic pressure, the osmolytes concentrations as well as the grain yield. Furthermore, the osmotic pressure of flag leaf sap appeared to depend on proline, TSN, TSS, organic acids and the ions content. The economic yield (grain yield) was positively correlated with proline, keto-acids and osmotic pressure but negatively correlated with TSN, TSS and citric acid.
Key words: (Triticum aestivum L.)/drought/glycine betaine/ osmolytes/salicylic acid/yield
List of abbreviations: Glycine betaine = GB, Salicylic acid = SA, Water stress = WS
Osmotic adjustment is the main component of physiological machinery, by which plants respond to soil water deficits (Chaves et al., 2003). Plants with higher osmotic regulators can absorb water from soil water
deficit condition. In wheat, many reports showed that wheat cultivars with higher K+, proline, soluble sugar at different growth stages performed better drought resistance (Dhanda et al., 2004). Furthermore, osmotic
adjustment results in an active accumulation of solutes within the plants in response to a lowering of soil water potential (WP) under water deficit (Turner and Jones, 1980). In general, OA is achieved by absorbing ions (e.g., K+, Na+, Ca2+, Mg2+, Cl", NO3" , SO4" and HPO4") or by accumulating organic solutes (e.g., free amino acids, sugar alcohols, quaternary ammonium compounds, and sugars) (Moinuddin et al., 2005). Waste water caused marked increases in OP, osmolytes [proline, organic acids, chloride and heavy metals (Cd++, Pb++, Cu++, Ni++ & Zn++)] content in flag leaves of wheat plants at heading and anthesis stages. On the other hand, waste water stress induced marked decreases in total soluble nitrogen (TSN), total soluble sugars (TSS) and ions (Na+, K+, & Ca++) as well as grain yield. The osmotic pressure appeared to depend mainly on proline, organic acids, chloride and heavy metals content, where there is positive correlations between OP and proline, organic acids, and heavy metals. The economic yield (grain yield) was positively correlated with TSN, TSS and ion contents but negatively correlated with proline, organic acids, chloride, heavy metals and OP (Aldesuquy et al., 2011).
Compatible osmolytes reported to be affected by drought stress include: proline, have improved plant tolerance to drought and salinity in a number of crops (Zhu et al., 2005). Carbohydrates, metallic ions, nitrogen and organic acids have been intensively studied in plants under drought conditions as osmolytes (Ashraf and Foolad, 2007; Lee et al., 2008 ). In addition, a positive relationship between osmotic adjustment OA and grain yield under water deficit has been shown in grain sorghum (Ibrahim et al., 1999), wheat (Blum et al., 1999), pea (Rodriguez-Maribona et al., 1992), and pigeonpea (Subbarao et al., 2000). A significant increase in seed yield of a group of genotypes with high OA over that with low OA has also been reported under water deficit condition in different crops (Moinuddin and Chopra, 2004).
Among wheat genotypes, significant differences regarding OA capacity exist and are associated with differences in crop growth and grain yield when water deficits in the soil and atmosphere are large enough to
cause substantial reductions in plant WPs (Moinuddin et al., 2005).
Glycine betaine (N,N,N-trimethyl glycine, GB), which is found in plants, animals, and bacteria (Prasad and Pardha-Saradhi, 2004) and many studies indicate that GB might play an important role in enhancing plant tolerance to some abiotic stresses such as salt, drought, and extreme temperatures (Quan et al., 2004). The
accumulation of endogenous GB is induced under stress conditions, and the levels are correlated with the extent of increased tolerance (Park et al., 2004). Significant advances have been made in alleviating the effects of environmental stresses by exogenously applied glycine btaine in different crops, such as wheat (Sayed et al., 2007), rice (Rahman et al., 2002), sorghum (Ibrahim and Aldesuquy 2003; Ibrahim, 2004) as well as sunflower (Iqbal et al., 2008).
Salicylic acid has received much attention due to its role in plant responses to abiotic stresses such as ozone (Koch et al., 2000), UV-B (Surplus et al., 1998), heat stress (Senaratna et al., 2000; Clark et al., 2004 ) drought (Nemeth et al., 2002; Singh and Usha, 2003), oxidative stress (Shim et al., 2003), salt and osmotic stress (Khodary, 2004; El-Tayeb, 2005; Arfan et al., 2007; Sawada et al., 2007), as well as the injurious action of heavy metals on rice plants (Mishra and Choudhuri, 1999); in sunflower (El-Tayeb et al., 2006).
The present work was undertaken to investigate the effect of foliar application of glycine betaine and grain presoaking in salicylic acid and their interaction on osmolytes in relation to osmotic adjustment and grain yield of droughted wheat (Triticum aestivum L.) cultivars by determining osmotic pressure, some organic solutes, ions and grain yield.
MATERIALS AND METHODS Plant material and growth conditions
Two wheat cultivars (Triticum aestivum L.) Sakha 94 (sensitive var.) and Sakha 93 (resistant var.) were used in this study. The variety Sakha 93 is known to be more drought resistant than Sakha 94.
A homogenous lot of wheat grains (i. e. either sensitive or resistant var.) was separately surface
sterilized by soaking in 0.01 % HgCl2 for 3 minutes, followed by thoroughly rinsing in sterile water. The sterilized grains from each variety were divided into two sets (= 500 g per set for each var.). Grains of the 1st and 2nd sets were separately soaked in distilled water or salicylic acid (0.05 M), respectively. In 20 November 2005, grains of each set were planted in plastic pots (fifteen grains per pot; 25cm width X 30cm height) filled with 6 kg mixture of soil (clay and sand = 2:1, v/v ). The pots were kept in a greenhouse, and the plants were subjected to natural day/night conditions (minimum /maximum air temperature and relative humidity were; 29.2/33.2 °C and 63/68 %, respectively). Irrigation to field capacity was carried out when soil water content had fallen to 60% of its initial value. Twenty days after planting, the plants were thinned to five uniform seedlings per pot.
On the day 65 (at the beginning of heading) after planting the pots of the 1st set was allocated to four groups (20 pots per each group) as follow: control (cont.), water stress (WS), glycine betaine control (GB.), glycine betaine + water stress (GB + WS). The 2nd set group was allocated as follow: salicylic acid control ( SA ), salicylic acid + water stress (SA+WS), control glycine betaine + salicylic acid (GB + SA) and glycine betaine + salicylic acid + water stress ( GB+SA+WS). For glycine betaine (10 mM) treatment, the plants were sprayed by glycine betaine 48 hrs before starting the stress period and weekly during the stress period.
Water deficit was imposed by withholding water at the reproductive stage for 30 days within two periods :on the day 65 from planting (heading stage) and the day 80 from planting (anthesis stage). Each droughted pot received 500 ml water at the end of 1st stress period. At the end of stress periods, rewatering to the field capacity was carried out. The undroughted (control) plants were irrigated to the field capacity during the stress period, and all plants were left to grow until grain maturation under normal irrigation with tap water. Samples were taken for measurements of osmotic pressure and other osmolytes at the end of each stress period before rewatering (i.e after 80 and 95 days from planting).
At the bud stage, 20 days from planting, (i.e., tillering stage) and before heading (i.e., at ear emergence) the plants received 35 kg N ha-1 as urea and 35 kg P ha-1 as potassium dihydrogen phosphate as fertilizers.
Monitoring the water status of the soil
Soil water content (SWC) at the end of the stress period was estimated by the destructive method as recommended by Ritchie et al., (1990).
Measurement of osmotic pressure
The osmotic pressure of flag leaf sap was measured by the cryoscopic method (Walter, 1949) and described by El-Sharkawi and Abdel-Rahman (1974).
Determination of total soluble sugars
Total soluble sugars was extracted and determined by anthrone method of Riazi et al.(1985) as modified by Ibrahim (1999).
Determination of the total Soluble Nitrogen
The total soluble nitrogen was determined by the conventional semimicromodification of Kjeldahl method (Pine, 1955).
Estimation of proline
The method adopted for estimation of proline was essentially that described by Bates et al (1973). Determination of keto acids
Keto acids was determined according to the method adopted by Friedemann and Haugen (1943). Determination of citric acid
The method adapted for estimation of citric acid was essentially that described by Snell and Snell (1949). Determination of some mineral ions
The extracts of the experimental plants were analyzed for the cations: Na+, K+ and Ca+2 Mg+2 measured by flam emission spectrophotometery (Williams and Twine 1960) and the anions Cl- chlorides were determined by the AgNO3 titration method as described by Hansen and Munns (1988).
Statistical analysis
The main effect of factors (watering regime, both used chemicals, growth stages and wheat type) and interaction (watering regime, both used chemicals, growth stages and wheat type) were evaluated by general linear model (two way ANOVA) using SPSS
program. Tests for significant differences between means at P =0.05 were given by LSD test. The correlation coefficient between the economic yield and all evaluated criteria was also evaluated.
RESULTS
Changes in soil moisture content
Soil moisture content was markedly reduced at the end of the stress period, and it was found to be about 23.3 and 4.6 % ( % of oven soil dry weight) for control and droughted pots respectively.
Changes in osmotic pressure
Water stress induced a noticeable increase (P<0.05) in osmotic pressure, as compared to control values, of wheat flag leaf of both cultivars at heading and anthesis stages. The resistant plants showed higher values osmotic pressure than the susceptible ones. Treatments with GB or SA caused significant increases (P<0.05) in the osmotic pressure of both wheat cultivars at heading and anthesis except GB with susceptible cultivar and SA with resistant one at heading stage. Furthermore, GB and SA treatment increased osmotic pressure more than the other treatments under controlled and stress conditions at heading and anthesis stages of both cultivars (Fig. 1a). The osmotic pressure appeared to be positively correlated with the osmolytes (proline, TSN, TSS, keto-acid and citric acid) and ions of the two wheat cultivars at heading (r =0.73-0.98) and at anthesis (r =0.49-0.79). Changes in proline
As compared to the control values, water stress caused non-significant increases in proline concentration in wheat flag leaf of both cultivars at heading and anthesis stages. The proline level of the resistant cultivar was higher than the sensitive one. In general, foliar application of GB or grain presoaking in SA caused significant increase (P<0.05) the proline concentrations in flag leaf of both wheat cultivars at heading and anthesis. Plants treated with GB+SA had higher proline concentrations than those treated with GB or SA only at heading and anthesis of the two wheat cultivars under controlled and water stress conditions (Fig.1b).
Changes in total soluble nitrogen (TSN)
In relation to control values, water stress induced a noticeable increase (P<0.05) in total soluble nitrogen of
wheat flag leaf of both cultivars at heading and anthesis stages, but the susceptible plants accumulated more soluble nitrogen than the resistant plants. In the majority of cases, treatments either with GB or SA caused additional increases in the TSN in flag leaf of both wheat varieties at heading and anthesis. Furthermore, GB and SA treatments increased (P<0.05) TSN concentration more than the other treatments under controlled and stress conditions of the two wheat varieties at heading and anthesis stages (Fig. 1c).
Changes in total soluble sugar (TSS)
Drought induced marked increases (P<0.05) in total soluble sugar of wheat flag leaf of both cultivars at heading and anthesis stages as compared to control values. It is clear that the susceptible plants accumulated more sugars than the resistant ones. In general, the applied chemicals GB or SA, increased (P<0.05) the TSS in flag leaf of both wheat cultivars at heading and anthesis under control and water stress conditions. In comparison to all treatments, the effect of GB+SA on TSS of wheat flag leaf of both cultivars was more effective (Fig. 1d ) Changes in keto-acids
In relation to control values, keto-acids accumulated in response to drought stress in wheat flag leaf of both cultivars at heading and anthesis stages and the resistant plants accumulated more keto-acids than the susceptible one. In general, GB or SA induced a marked increases (P<0.05) in the keto-acids concentration in flag leaf of both wheat varieties at heading and anthesis under control and water stress conditions. The effect was more pronounced with GB+SA treatment (Fig. 2a).
Changes in citric acid
Water stress caused a marked increase (P<0.05) in the citric acid in wheat flag leaf of both cultivars at heading and anthesis stages and the resistant plants accumulated more citric acid than the susceptible ones. GB, SA or their interaction caused a noticeable increases (P<0.05) in the citric acid concentration under water stress of both cultivars at heading and anthesis stages (Fig. 2b).
Changes in ions content
In relation to control values, water stress induced
significant increases (P<0.05) in ions content (Na+, K+, Ca+2, Mg+2, and Cl- ) as well as Na+/K+ ratio in wheat flag leaf of both cultivars at heading and anthesis stages. On the other hand, the resistant plants accumulated more ions than the susceptible ones. In general, GB or SA caused an additional increase (P<0.05) in the ions content under controlled and water stress conditions and this effect was more pronounced with GB and SA treatment (Fig. 2c, 2d, 3a, 3b, 3c and 3d).
Changes in grain yield
Water stress reduced (P<0.05) the grain yield of both wheat cultivars. This effect was more pronounced with sensitive plants. The used chemicals improved the grain
yield of both cultivars. Glycine betaine + salicylic acid treatments appeared to mitigate the effect of water stress on wheat plants more than the other treatments under control and stress conditions (Fig. 4).
The economic yield (grain yield) appeared to be positively correlated with proline, keto-acids and osmotic pressure (r= 0.48-0.77) for the sensitive wheat variety and (r = 0.36-0.7) for the resistant one. However, grain yield was negatively correlated with total soluble nitrogen, total soluble sugars and citric acid (r = -0.080.32) for the sensitive wheat variety and (r = -0.18 -0.39) for the resistant one.
Figure 1. Effect of glycine betaine, salicylic acid and their interaction on osmotic pressure (atm.) proline, total soluble nitrogen and total soluble sugars (mg g-1 dwt) in flag leaf sap of stressed wheat cultivars at heading and anthesis. Vertical bars represent LSD values at P< 0.05.
Figure 2. Effect of glycine betaine, salicylic acid and their interaction on keto-acids, citric acid (mg g-1 dwt), Na+ and K+ (mmol g-1 dwt) in flag leaf sap of water stressed wheat cultivars at heading and anthesis. Vertical bars represent LSD values at P< 0.05.
Figure 3. Effect of glycine betaine, salicylic acid and their interaction on Ca+2, Mg+2, Cl- (m mol g-1dwt) and Na+/K+ in flag leaf sap of water stressed wheat cultivars at heading and anthesis. Vertical bars represent LSD values at P< 0.05.
Figure 4. Effect of glycine betaine, salicylic acid and their interaction on grain yield of wheat cultivars grown under water stress condition. Vertical bars represent LSD values at P< 0.05.
turgor is necessary for maintance of growth through cell
DISCUSSION
In order to understand the physiological adaptation of Triticum aestivum to stress induced by water stress osmotic pressure, proline, TSN, TSS, organic acids and ions content in flag leaf sap were studied particularly after ear emergence (at heading) and at the beginning of the grain set (at anthesis). Thus, the results showed that water stress induced a marked increase in osmotic pressure. This is probably due to the increasing in proline, TSN, TSS, organic acids and ions content. Furthermore, osmotic pressure appeared to be positively correlated with organic osmolytes as well as inorganic acids. In accordance to these results, Bulm (1996) concluded that osmolytes accumulation (OA) in plant cells results in a decrease of the cell osmotic potential and thus in maintenance of water absorption and cell turgor pressure, which might contribute to sustain physiological processes, such as stomatal opening, photosynthesis, and growth expansion. Furthermore, occurrence of OA at sensitive crop reproductive stages has been reported to play a constructive role against floral abortion (Wright et al., 1983), which results in maintaining grain number under water deficit (Moinuddin and Chopra, 2004). Additionally, OA has also been claimed to facilitate a better translocation of preanthesis carbohydrate reserves to the grain during the grain-filling period (Subbarao et al., 2000).
Solutes accumulation in plant cells creates an intracellular osmotic potential which in the presence of rigid cell wall generates turgor pressure. Maintaince of
elongation (Yancey et al., 1982). Moreover, The
accumulation of these solutes may not be important for osmotic stress tolerance but the metabolic pathways may have adaptive value (Hasegawa et al., 2000). A further hypothesis is that compatible solutes are also involved in scavenging reactive oxygen species (Chen and Murata, 2002).
Aldesuquy et al. (2012) reported that seawater concentrations (10, 25%) caused noticeable increase in osmotic adjustment, organic solutes (TSS, TSN, proline, organic acids and glycerol) and inorganic ions (Na+, K+, Ca2+, Mg2+ and Cl") in two wheat cultivars. On the other hand, clear reduction in K/ Na ratio in the flag leaves of both cultivars was observed. The capacity of osmotic adjustment was greater in younger leaves than in older ones particularly with higher concentration (25%) in both cultivars. Osmotic pressure of flag leaf sap appeared to depend mainly on proline, TSN, TSS, organic acids, glycerol and ions content, where there is a positive correlation between osmotic pressure and all of them.
The present results indicated that GB, SA or their interaction increased the measured osmotic pressure and osmolytes concentration ( proline, total soluble nitrogen, total soluble sugars, ions content (Na+, K+, Mg+2, Ca+2 and Cl" ) as well as Na+/K+ in wheat plants subjected to water deficit. In fact, GB added more increase to wheat osmotic pressure and these results are similar to those obtained by Ibrahim (2004) with sorghum plants grown under salinity stress. In this respect, Iqbal et al. (2008) found that water stress significantly decreased leaf water
contents, osmotic and turgor potentials in two sunflower lines and foliar application of GB at the vegetative or reproductive growth stage increased leaf water and turgor potentials to some extent in both sunflower lines when grown under water stress. Moreover, Sulian et al.
(2007) suggested that GB may not only protect the integrity of the cell membrane from drought stress damage, but also be involved in OA in transgenic cotton plants. Salicylic acid caused an additional increase in wheat osmotic pressure as well as the studied osmolytes and these are the same results obtained by Chinnusamy and Zhu (2003).
Proline content of the flag leaves increased at both stages of growth in both wheat cultivars under water stress condition. The accumulation of proline, primarily in the cytosol, often occurs in plants under stress with strong correlation between stress tolerance and proline accumulation, but the relationship is not universal and may be species dependent (Ashraf and Foolad, 2007). There is other roles were proposed for proline besides osmotic adjustment in stressed plants include acting as hydroxyl scavenger, stabilization of membrane and protein structure, as a sink for carbon and nitrogen for stress recovery, and buffering cellular redox potential under stress (Lee et al., 2008). Moreover, high levels of proline enabled the plant to maintain low water potentials (Sankar et al., 2007). By lowering water potentials, the accumulation of compatible osmolytes, involved in osmoregulation allows additional water to be taken up from the environment, thus buffering the immediate effect of water shortages within the organism (Kumar et al., 2003).
It is important to note that proline accumulated with GB application in the two wheat cultivars under water stress compared to drought-stressed plants alone suggests that proline accumulation together with GB in leaves presumably decreased the extent of drought-induced damage even more than drought-stressed group alone. The protective role of GB was more pronounced in the sensitive cv. Therefore, exogenous GB application might be a useful method to improve growth and productivity of higher plants and retard aging process
under drought-stressed conditions.
An increase in proline concentration in SA-treated plants under normal and stress conditions was also observed. Proline can thus be considered as an important component in the spectra of SA-induced ABA-mediated protective reactions of wheat plants in response to drought, contributing to a reduction in the injurious effects of drought and an acceleration of the reparation processes following stress, evidencing the protective action of SA on wheat plants (Shakirova, 2003). .
In the present investigation the TSN accumulated in response to water stress conditions in both wheat cultivars. Such accumulation may be mainly resulted from a sharp increase in total free amino acids, total soluble proteins and glycine betaine (Ibrahim, 2004). This change in nitrogen content may be related to the inhibition of translocation from root to shoot, inhibition of protein synthesis or the increase in protease activity (Khalil and Mandurah, 1990). The increase in the soluble nitrogen compounds are of importance in plant osmoregulation in response to water deficit. The application of GB, SA or their interaction caused an accumulation in TSN in both wheat cultivars at heading and anthesis.
Our findings indicate that water stress increased TSS in both wheat cultivars. This may result from increased starch hydrolysis, synthesis by other pathways or decreased conversion to other products. Several studies have shown an increase in amylase activity in water-stressed leaves (e.g. Keller and Ludlow, 1993). Alternatively, increased translocation of carbohydrates into leaves or a decrease in translocation of carbohydrates from leaves could also contribute to the observed sugar accumulation. In this respect Xue et al. (2008) found that water deficit in wheat leaves caused a reduction in photosynthesis and high demands for osmolyte synthesis especially total soluble sugars.
The marked increase in the content of soluble sugars in flag leaf sap of stressed and nonstressed plants treated with GB, SA or their interaction agrees with the results obtained by El Tayeb (2005) who recorded an additional increase in Na, soluble proteins and soluble sugars in salt-
stressed barley grains due to application of SA. SA treatments increased K+/Na+ ratio in the plant leaves under drought condition.
Organic acid content (citric acid and keto-acids) were increased in both wheat cultivars after withholding water. The increase in organic acids content may be a result of drought induced synthesis (Venekamp et al., 1989) and it is important for plant osmotic adjustment under water stress (Morgan, 1984), and regulation of pH of plant cells (Venekamp et al., 1989). These results were in a good conformity with those obtained by many others who recorded the increase in organic acids in response to water stress (Ibrahim et al., 1999) and salinity (Shi and Sheng, 2005). Also, Hasaneen et al., (1990) found that a-ketoglutaric and some carboxylic acids of Krebs cycle increased in Zea mays seedlings and plants in response to salinity stress.
The application of GB, SA or their interaction caused an accumulation in organic acids in both wheat cultivars at heading and anthesis. The dramatic increase in the organic acids in response to GB, SA or their interaction may probably be due to the importance of organic acids in plants osmotic adjustment under water stress (Morgan,1984). Moreover, increased concentration of organic acids may involve in oxidative respiration originate from enhanced synthesis induced by dehydration and are directly linked to the proline
synthesis pathway, a mechanism which control the
cytoplasmic pH level (Venekamp et al., 1989).
An important results of the present study is that water stress, in the absence of salinity in the root zone, induced a conspicuous increase in the flag leaves ions
(Na+, K+, Mg+2, Ca+2 and Cl- ) as well as Na+/K+
concentrations. The effects of both stresses (water and salt stress) are not strictly additive in reducing plant performance and that tolerances to water and salt stress are linked through a common mechanism of Na uptake for osmotic adjustment (Glenn and Brown, 1998). A plant in drying soils is exposed to increasing levels of both water deficit and osmotic stress because the soil matrix potential decreases simultaneously with decreasing soil moisture. Even if osmotic adjustment occurs, a decrease
in the hydraulic conductivity of root membrane is observed and it has been clearly demonstrated that the impact of Na+ on water-channel function is not due to its osmotic effect (Carvajal et al., 2000).
Another role of Na in C3 species such as wheat is related to its involvement in photosynthesis (Brownell and Bielig, 1996). An increased sodium concentration in plants experiencing water stress may be related to an increase in the metabolic requirement of sodium to sustain photosynthesis in these conditions. Sodium has been reported to be involved in the maintenance of mesophyll chloroplast structure, mainly in relation to granal stacking (Brownell and Bielig, 1996), and thus sodium deficient C3 plants exhibit a wide range of chlorophyll a fluorescence perturbations (Grof et al., 1986). Sodium may also be involved in the regeneration of phosphoenolpyruvate in mesophyll chloroplasts (Murata et al. 1992) because a Na+ gradient across the envelope could be an alternative energy source for the active transport of pyruvate (Ohnishi et al., 1990). An increase in sodium uptake could then be the consequence of a stress-induced decrease in the efficiency of the Na +/pyruvate co transport-system. This confirms the previous findings of Martinez et al. (2003) who reported that drought-induced specific increase in Na+ concentration in two populations of Atriplex halimus under water stress.
The application of GB, SA or their interaction caused an accumulation in Na+ in both wheat cultivars at heading and anthesis. This is in agreement with Ibrahim (2004) who studied the efficacy of exogenous glycine betaine application on sorghum plants grown under salinity stress. Salinity reduced sorghum growth. Foliar application of glycine betaine at 75 mM mitigated the adverse effects of salinity. The measured osmotic pressure and solutes concentration (Na, K, Ca, Mg, total soluble sugar and betaine) increased while Na+/K+ ratio decreased in sorghum plants. Glycine betaine added more increase to sorghum osmotic pressure and increased the concentration of soluble sugars
Potassium content was increased to some extent in both wheat cultivars at heading and anthesis, higher K+
concentrations, effectively stabilize native protein (Borowitzka, 1981).This had been observed by (Ibrahim, 1999). Moreover, the drought resistant cultivar had a higher K+ concentrations in the face of drought stress more than the sensitive variety and this reflects the important role of this cation in wheat plants adaptation to water stress conditions.
The application of GB, SA or their interaction caused an accumulation in K+ in both wheat cultivars at heading and anthesis. In this respect, Gunes et al. (2005) demonstrated that SA treatments caused N accumulation in plants and increased P, K, Mg and Mn concentrations under stress conditions. On the other hand Hamada and Al-Hakimi (2001) observed positive effects of SA in the Na, K, Ca and Mg content of wheat plants grown under salinity. SA application inhibited Na accumulation in salinity condition. Thus seed pretreatment with SA induced a reduction in sodium absorption and toxicity, which was further reflected in low membranes injury, high water content and dry matter production.
Our data showed, that calcium ion accumulated in response to water stress. Ca2+ plays important roles in plant responses to drought resistance. Nayyar (2003) also found in wheat that Ca2+ appeared to reduce the devastating effects of stress by elevating the content of proline and glycine betaine, thus improving the water status and growth of seedlings and minimizing the injury to membranes. In fact, Calcium ion has unique properties and universal ability to transmit diverse signals that trigger primary physiological actions in cells in response to hormones, pathogens, light, gravity, and stress factors. Being a second messenger of paramount significance, calcium is required at almost all stages of plant growth and development, playing a fundamental role in regulating polar growth of cells and tissues and participating in plant adaptation to various stress factors. Many researches showed that calcium signals decoding elements are involved in ABA-induced stomatal closure and plant adaptation to drought, cold, salt and other abiotic stresses and some new studies show that Ca2+ is dissolved in water in the apoplast and transported primarily from root to shoot through the transpiration
stream (Song et al., 2008).
Glycine betaine, salicylic acid or their interaction treatments added more increases in Ca+2 content in wheat cultivars. These results were in a good agreement with Sayed et al. (2007) who reported higher increases in GB, K+, Ca+2 and Na+/K+ ratio in two cultivars of wheat (tolerance and salt sensitive) treated with GB under salt stress. High accumulation of GB and K+ mainly contributed to osmotic adjustment, which is one of the factors known to be responsible for improving growth and yield under salt stress.
Magnesium content was increased to some extent in both wheat varieties at heading and anthesis, this increase was also demonstrated by Ibrahim (1999) who reported that drought stress favors the accumulation of Mg+2 in plants. On the other hand the applied chemicals added more increase in the Mg+2 of wheat cultivars. These are the same result obtained by Ibrahim (2004).
Perusal data showed that grain yield was reduced by water stress condition. In fact yield is a result of the integration reactions in the plants, consequently any factor that influences this metabolic activity at any period of plant growth can affect the yield (Ibrahim et al.,1999). These were the same results obtained by Ali et al. (2007) with wheat plants under water stress. In this respect, Shao et al. (2008) state that, seed yield and yield components are severely affected by water deficit. Water stress reduced the head diameter, 100-achene weight and yield per plant in sun flower (Blumwald et al., 2004). These authors also observed significant but negative correlation of head diameter with fresh root and shoot weight under water stress. A positive and significant relation was recorded between dry shoot weight and achene yield per plant. Reddy et al. (1998) supported the view that water stress for more than 12 days at the grain-filling and flowering stage of sunflower (grown in sandy loam soil ) was most damaging and reduced the achene yield.
In this respect, Morgan (1995) reported comparisons of wheat grain yield under drought for low and high osmotic-adjusting lines. Among the nine comparisons
presented, only three pairs had a significantly greater yield in high osmotic adjustment lines, and these were limited to cases of severe water deficits and very low grain yield. Three other comparison pairs had small nonsignificant advantage for high osmotic-adjusting lines, and three pairs had lower although non-significant yield for the high osmotic-adjusting group. Furthermore, Moinuddin et al. (2005) investigated the substantial differences in OA among the bread wheat genotypes. A high positive correlation (P<0.001) between OA of greenhouse-stressed plants and the grain yield recorded in the field under water deficit indicates that improved bread wheat cultivars could be successfully screened for drought environments in the greenhouse on the basis of OA. Besides, a significant (P<0.05) correlation between OA and grain yield under simulated water deficit condition in the field further advocates that OA could be regarded as a selection criterion for screening improved bread wheat genotypes for water deficit environments. Moreover, this study also indicates that OA is a heritable character and plays an important role in maintaining yield stability under water deficit.
The applied chemicals appeared to mitigate the effect of water stress on wheat yield and the effect was more pronounced with (GB+SA) treatment. This improvement would result from the beneficial effect of the provided chemicals on growth and metabolism of wheat plants under water deficit conditions. These results were in agreement with the results obtained by Iqbal et al.
(2008). In addition, Arfan et al. (2007) investigated the improvement in growth and grain yield of wheat salt-tolerance due to SA application. Shakirova et al. (2003) reported that, the treatment of wheat plants with 0.05 mM salicylic acid (SA) increased the level of cell division within the apical meristem of seedling roots causing an increase in plant growth and an elevated wheat productivity.
Finally, we can conclude that the interactive effect of glycine betaine + salicylic acid represent the most effective treatment in improvement the drought tolerance of sensitive wheat cultivars by increasing the osmolytes and consequently increase the osmotic
adjustment and grain yield.
REFERENCES
Ali, M. H., Hoque, M.R., Hassan, A. A. and Khair, A. (2007) Effects of deficit irrigation on yield, water productivity, and economic returns of wheat. Agriculture Water Management 92, 151 - 161.
Arfan, M., Athar, H. R. and Ashraf, M. (2007) Does exogenous application of salicylic acid through the rooting medium modulate growth and photosynthetic capacity in two differently adapted spring wheat cultivars under salt stress? Journal of Plant Physiology 164, 685-
694.
Ashraf, M. and Foolad, M.R. (2007) Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environmental and Experimental Botany 59, 206-216.
Bates, L. S., Waldren, R.P. and Teare, I.D. (1973) Rapid determination of free proline for water stress studies. Plant and soil 39, 205-207.
Blum, A. (1996) Crop response to drought and the interpretation of adaptation. Plant Growth Regulation 20, 135-148.
Aldesuquy, H.S., Baka, Z.A.M, El-Shehaby, O.A. and Ghanem, H.E. (2012) Efficacy of seawater salinity on osmotic adjustment and solutes allocation in wheat (Triticum aestivum) flag leaf during grain filling. International Journal of Plant Physiology and Biochemistry 4, 33-45.
Aldesuquy, H.S., Haroun, S.A., Abo-Hamed, S.A. and Elsaied, A.A. (2011) Physiological studies of some polyamines on wheat plants irrigated with waste water. Osmolytes in relation to osmotic adjustment and grain yield. Phyton 50, 263-268.
Blum, A., Zhang, J. and Nguyen, H.T. (1999)
Consistent differences among wheat cultivars in osmotic adjustment and their relationship to plant production. Field Crops Research 64, 287291.
Blumwald, E., Anil, G. And Allen, G. (2004) Effect of water stress on growth, Na+ and K+ accumulation and water use efficiency in relation to osmotic adjustment in two populations of Atriplex halimus L. Plant Growth Regulation 41, 63-73.
Borowitzka L, J. (1981) Soluble accumulation and regulation of cell water activity: L. G. Paleg and
D. Aspinall (Eds): The physiology and
biochemistry of drought resistant in plants . Academic Press: Syndey, 97-130.
Brownell P.F. and Bielig L.M. (1996) The role of sodium in the conversion of pyruvate to phosphoenolpyruvate in the mesophyll chloroplasts of C plants. Aust. J. Plant Physiol. 23, 171-177.
Carvajal, M., Cerda, A. and Martinez, V. (2000) Does calcium ameliorate the negative effect of NaCl on melon root water transport by regulating aquaporin activity? New Phytolology 145, 439447.
Chaves, M.M, Maroco, J.P. and Periera, S. (2003) Understanding plant responses to drought from genes to the whole plant. Function Plant Biology 30, 239-264.
Chen, T. H. and Murata, N. (2002) Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr. Opin. Plant Biology 5, 250-257.
Chinnusamy, V. and Zhu, J.K. (2003): Topics in current genetics. In: H. Hirt, K. Shinozaki (Eds.): Plant stress responses to abiotic stress. Springer-Verlag Berlin Heidelberg: 4.
Clark, S.M., Mur, L.A. J., Wood, J.E. and Scott I.M.
(2004) Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana. Plant Journal 38, 432437.
Decheng, S and Yanmin, S (2005) Effect of various salt-alkaline mixed stress conditions on sunflower seedlings and analysis of their stress factors. Environmental and experimental Botany 54, 8-21.
Dhanda, S., Sethi, G.S. and Behl, R.K. (2004) Indices of drought tolerance in wheat genotypes at early stages of plant growth, Journal of Agronomy & Crop Science 190, 6-12.
El-Tayeb, M. A. (2005) Response of barley grains to the interactive effect of salinity and salicylic acid. Plant Growth Regulation 45, 215-224.
El-Tayeb, M. A., El-Enany, A.E. and Ahmed, N.L.
(2006) Salicylic acid induced adaptive response to copper stress in sunflower (Helianthus annuus L.). Plant Growth Regulation 50, 191199.
El-Shakawi, H. M. and Abdel-Rahman, A.A. (1974) Response of olive and almond orchards to partial irrigation under dry farming practices in semi arid regions. II- Plant soil water relations in olive during the growing season. Plant and soil (Netherland) 41, 13-32.
Friedemann, T.E. and Haugen, G.E. (1943) Pyruvic acid. II. The determination of keto acids in blood and urine. Journal of Biologycal Chemistry 147, 415-442.
Kumar, S.G., Reddy, A.M. and Sudhakar, C. (2003) NaCl effects on proline metabolism in two high yielding genotypes of mulberry (Morus alba L.) with contrasting salt tolerance. Plant Science
165, 1245-1251.
Glenn, E.P. and Brown, J.J. (1998) Effects of soil salt levels on the growth and water use efficiency of Atriplex canescens (Chenopodiaceae) varieties in drying soil. American Journal of Botany 85, 10- 16.
Grof, C.P.L., Johnston, M. and Brownell, P.F. (1986) In vivo chlorophyll a fluorescence in sodium-deficient C plants. Australlian Journal of Plant Physiology 13, 589-595.
Gune§, A., inal, A., Alpaslan, M., Qifek, N., Guneri,
E., Eraslan, F. and Guzelordu, T. (2005): Effects of exogenously applied salicylic acid on the induction of multiple stress tolerance and mineral nutrition in maize (Zea mays L.). Archives of Agronomy and Soil Science 51, 687 -
695.
Hamada, A.M. and Al-Hakimi, A.M.A. (2001): Salicylic acid versus salinity-drought-induced stress on wheat seedlings. Rostlinna Vyroba 47, 444-450.
Hansen, J.L and Munns, D.N. (1988) Effect of CaSO4 and NaCl on mineral content of Leucaena leucocephala. Plant and Soil 54, 101-107.
Hasaneen, M. N. A, Younis, M. E. and El-Saht, H. M. (1990) Plant growth, metabolism and adaptation in relation to stress conditions XII. Carbohydrates and acid accumulation in Phaseolus vulgaris and Zea mays stressed with sodium sulphate. Journal of Qatar University Science Bulletin 10: 185-197.
Hasegawa, P.M., Bressan, R.A., Zhu, J and Bohnert, H.J. (2000) Plant cellular and molecular responses to high salinity. Annual. Review. Plant Molecular Biology 51, 463-499.
Ibrahim, A.H. (1999) Control of growth of sorghum plants grown under stress conditions. Ph. D
Thesis, Faculty of Science, Mansoura University, Egypt.
Ibrahim, A.H. (2004) Efficacy of exogenous glycine betaine application on sorghum plants grown under salinity stress. Acta Botanica Hungarica,
46, 307-318.
Ibrahim, A.H. and Aldesuquy, H.S. (2003) Glycine betaine and shiki- mic acid induced modification in growth criteria, water relation and productivity of droughted Sorghum bicolor plants. Phyton (Horn, Austria) 43, 351-363.
Iqbal, N., Ashraf, M., Ashraf, M.Y. (2008) Glycinebetaine, an osmolyte of interest to improve water stress tolerance in sunflower (Helianthus annuus L.): water relations and yield. South African Journal of Botany 74, 274281
Keller, F. and Ludlow, M.M. (1993) Carbohydrate metabolism in drought-stressed leaves of pigeonpea (Cajanus cajan). Journal of Experimental Botany 44, 1351-1359.
Khalil, S. and Mandorah, H. M. (1990) Effects of water stress deficiency and kinetin on growth and nitrogen metabolism of cowpea plants. Journal of Agronomy and Crop Science 164, 9399.
Koch, J.R., Creelman, R.A., Eshita, S.M., Seskar, M., Mullet, J.E. and Davis, K.R.. (2000) Ozone sensitivity in hybrid poplar correlates with insensitivity to both salicylic acid and jasmonic acid. The role of programmed cell death in lesion formation. Plant Physiology 123, 487496.
Martinez, J.P., Ledent, J.F., Bajji, M., Kinet, J.M. and Lutts, S. (2003) Effect of water stress on growth, Na+ and K+ accumulation and water use efficiency in relation to osmotic adjustment in
two population of Atriplex halimus L. Plant Growth Regulation 41, 63-73.
Mishra, A. and Choudhuri, M.A (1999) Effect of salicylic acid on heavy metal-induced membrane deterioration mediated by lipoxygenase in rice. Biologia Plantarum 42, 409-415.
Moinuddin and Khanna-Chopra, R. (2004) Osmotic adjustment in chickpea in relation to seed yield and yield parameters. Crop Science 44, 449455.
Moinuddin, Fischer, R. A., Sayre, K.D. and Reynolds, M. P. (2005) Osmotic Adjustment in wheat in relation to grain yield under water deficit. Environments Agronomy. Journal 97, 10621071
Morgan, J. (1984) Osmoregulation and water stress in higher plants. Annual Review of Plant Physiology 35, 299-319.
Morgan, J.M. (1995) Growth and yield of wheat lines with differing osmoregulative capacity at high soil water deficit in season of varying evaporative demand. Field Crop Research 40, 143- 152.
Murata, S., Kobayashi, M., Matoh,T. and Sekiya, J. (1992) Sodium stimulates regeneration of phosphoenolpyruvate in mesophyll chloroplasts of Amaranthus tricolor. Plant Cell Physiology 33, 1247-1250.
Nayyar, H. (2003) Variation in osmoregulation in differentially drought-sensitive wheat genotypes involves calcium. Biologia Plantarum
47, 541-547.
Nemeth, M, Janda, T, Horvath, E, Paldi, E and Szalai,
G. (2002) Exogenous salicylic acid increases polyamine content but may decrease drought tolerance in maize. Plant Science 162, 569-574.
Ohnishi, J., Flugge, U., Heldt, H.W. and Kanai, R. (1990) Involvement of Na+ in active uptake of pyruvate in mesophyll chloroplasts of some C4 plants. Na+ /pyruvate cotransport. Plant Physiology 94: 950-959.
Park, E.J., Jeknic, Z. and Sakamoto, A (2004) Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage. The Plant Journal 40, 474-487.
Pine, N.W. (1955) Proteins. In: (K. Peack, and M. V. Traceyeds). Modem methods of plant analysis. IV, 23, Springer Verlage, Berlin.
Prasad, K and Pardha-Saradhi, P. (2004) Enhanced tolerance to photoinhibition in transgenic plants through targeting of glycinebetaine biosynthesis into the chloroplasts. Plant Science 166, 1197-1212.
Quan, R.D., Shang, M., Zhang, H. (2004) Improved chilling tolerance by transformation with betA gene for the enhancement of glycinebetaine synthesis in maize. Plant Science 166, 141-149.
Rahman, M.S., Miyake, H. and Takeoka, Y. (2002) Effects of exogenous glycine-betaine on growth and ultra-structure of salt-stressed rice seedlings (Oryza sativa L.). Plant Production Science 5, 33-44.
Riazi, A., Matsuda, K. and Arslan, A. (1985) Water stress induced changes in concentrations of proline and other solutes in growing regions of young barely leaves. Journal of Experimental Botany 36: 1716-1725.
Ritchie, S.W., Nguyen, H.T. and Holaday, A. S. (1990) Leaf water content and gas exchange parameters of two wheat genotypes differing in drought resistance. Crop Science 30, 105-111.
Rodriguez-Maribona, B., Tenorio, J.L., Conde, J.R.
and Ayerbe, L. (1992) Correlation between yield and osmotic adjustment of peas (Pisum sativum L.) under drought stress. Field Crops Research 29, 15-22.
Sankar, B., Jaleel, C.A., Manivannan, P., Kishorekumar, A, Somasundaram, R and Panneerselvam, R. (2007) Drought induced biochemical modifications and proline metabolism in Abelmoschus esculentus (L.) Moench, Acta Botanica Croatia 66, 43-56.
Sawada, H., Kim, D.W., Kobayashi, K. and Shim, I. S. (2007) In abenfide-induced alleviation of salt stress in rice as linked to changes in salicylic acid content and catalase activity, Journal of Crop Science Biotechnology 10, 41-46.
Sayed, H.R.,. Athar, H.A., Ashraf, M. and Hameed, A. (2007) Glycine betaine-induced modulation of antioxidant enzymes activities and ion accumulation in two wheat cultivars differing in salt tolerance. Environmental and Experimental Botany 60, 368-376.
Senaratna, T., Touchell, D., Bunn, E. and Dixon, K., (2000) Acetyl salicylic acid (Aspirin) and salicylic acid induce multiple stress tolerance in bean and tomato plant. Plant Growth Regulation 30, 157-161.
Shakirova, F.M., Sakhabutdinova, A.R, Bezrukova, M.V., Fatkhutdinova, R.A. and Fatkhutdinova, D.R. (2003) Changes in the hormonal status of wheat seedlings induced by salicylic acid and salinity. Plant Science 164, 317-322.
Shao, HB, Chu, LY, Jaleel, CA and Zhao, CX. (2008) Water deficit-induced morphological changes in higher plants. C. R .Biology 331, 215 -225.
Shim, I.S., Momose, Y., Yamamoto, A., Kim, D.W. and Usui, K. (2003) Inhibition of catalase activity by oxidative stress and its relationship
to salicylic acid accumulation in plants. Plant Growth Regulation 39, 285-292.
Singh, B. and Usha, K. (2003) Salicylic acid induced physiological and biochemical changes in wheat seedlings under water stress, Plant Growth Regulation 39, 137- 141.
Snell, F. D. and Snell, C. T. (1949) Colorimetric methods of analysis. Volume II. New York, Van Nostrand.
Subbarao, G.V., Nam, N.H., Chauhan, Y.S. and Johansen, C. (2000) Osmotic adjustment, water relations and carbohydrate remobilization in pigeonpea under water deficits. Journal of Plant Physiol. 157,651-659.
Sulian, Lv, Yan, A., Zhang, K., Wang, L. and Zhang, J.
(2007) Increase of glycinebetaine synthesis improves drought tolerance in cotton. Molecular Breeding 20, 233-248.
Surplus, S.L., Jordan, B.R., Murphy, A.M., Carr, J.P, Thomas, B. and Mackerness, S.A.H. (1998) Ultraviolet-B-induced responses in Arabidopsis thaliana: role of salicylic acid and reactive oxygen species in the regulation of transcripts encoding photosynthetic and acidic pathogenesis-related proteins. Plant Cell Environment 21, 685-694.
Lee, V.G., Robert, N. C., Ronny, R. D., Mark, A. E. and Mark, W. R. (2008) Synthesis of organic osmolytes and salt tolerance mechanisms in Paspalum. Environmental and Experimental Botany 63, 19-27.
Venekamp, J H, Lamp, J E and Koot, J T (1989) Organic acids as sources for drought induced proline synthesis in field bean plants Vicia vaba L. Journal of Plant Physiology 133, 654-659.
Walter, H. (1949) Gundlagen der flanzen verlientung. Eintubring, in die
pflanzengeographie- fur studierends der hocholen, standorstlehre.: Stuttgart, Ulmer.
Song, W.Y, Zheng-Bin, Z., Shao, H.B, Guo, Z.L., Cao,
H.X., Zhao, H.B., Fu, Z.Y. and Hu, X.J. (2008) Relationship between calcium decoding elements and plant abiotic-stress resistance. International. Journal of Biological Science 4, 116-125.
Williams, V. and Twine, S. (1960) Flam photometer method for sodium, potassium and calcium. In: K. Peach and M. V. Tracey (Eds) . Modern Methods of Plant Analysis. . Springer- Verlag, Berlin Vol. V. pp 3-5.
Wright, G.C., Smith, R.C.G. and Morgan, J.M. (1983) Differences between two grain sorghum genotypes in adaptation to drought stress: III.
Physiological responses. Australlian Journal of Agricultural Research 34, 637-651.
Xue, G. P., McIntyre, C. L., Glassop, D.and Shorter, R
(2008) Use of expression analysis to dissect alterations in carbohydrate metabolism in wheat leaves during drought stress. Plant Molecular Biology 67, 197-214.
Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D. and Somero, G. N. (1982) Living with water stress: Evolution of osmolyte systems. Science 217, 1214-1222.
Zhu, X, Gong, H, Chen, G, Wang, S and Zhang, C.
(2005) Different solute levels in two spring wheat cultivars induced by progressive field water stress at different developmental stages. Journal of Arid Environmental 62: 1-14.
