Art08_Nudrat.indd Journal of Applied Botany and Food Quality 84, 169 - 177 (2011) 1Department of Botany, University of Agriculture, Faisalabad, Pakistan *2Second affi liation: Department of Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia Does exogenous application of salicylic acid improve growth and some key physiological attributes in sunfl ower plants subjected to salt stress? Sibgha Noreen1, Muhammad Ashraf 1,2, Nudrat Aisha Akram*1 (Received September 2, 2010) * Corresponding author Summary To appraise the effect of foliar-applied salicylic acid (SA) on growth and some key physiological attributes in sunfl ower plants under salt stress, a greenhouse experiment was conducted. Two sunfl ower lines (Hysun-33 and SF-187) were subjected to non-saline (control) and saline regimes (120 mM NaCl). After 14 days of initiation of salt treatment plants of both sunfl ower lines were supplied with varying levels (100, 200 and 300 mg L-1) of SA applied foliarly to all sunfl ower plants exposed to normal or saline substrates. After 21 days of SA application, data for growth (shoot biomass), photosynthetic pigments (chlorophyll a and b), water relation components, and accumulation of proline and mineral nutrients were recorded. Salt stress adversely affected the growth, chlorophyll pigments, water relations and contents of some key mineral nutrients, while increased the amount of proline and leaf and root Na+ as well as Cl- contents in both sunfl ower lines. Foliar-applied SA improved growth, chlorophyll a and b pigments, leaf turgor potential, and leaf and root Ca2+ concentrations. Of all salicylic acid levels used in the present study, 200 and 300 mg L-1 were found to be relatively more effective than the other levels in improving chlorophyll a and b pigments, leaf turgor potential, leaf and root Ca2+ concentration, while the other attributes remained unaffected due to SA application. Of both sunfl ower lines, Hysun-33 had higher amounts of photosynthetic pigments and essential nutrients than did SF-187. Introduction Plants exposed to saline environments experience four basic problems such as reduced water potential, accumulation of toxic ions (Na+, Cl-) and their harmful effects on various physiological and biochemical processes of the plant, thereby decreasing the absorption of some essential mineral nutrients such as K+ and Ca2+, hormone imbalance, and production of reactive oxygen species (ROS) (MUNNS, 2005; ASHRAF, 2009; ASHRAF et al., 2010). Availability of nutrients to plants depends on the functioning of membrane transporters that mediate the translocation of nutrients from the soil into the plant thereby compartmentalizing them into the cells, tissue and organs (TESTER and DAVENPORT, 2003; EPSTEIN and BLOOM, 2005). For example, Ca2+-ATPases (Ca2+-pumps) regulate low cytoplasmic Ca2+ levels in plants (GEISLER et al., 2000). Salt tolerant cultivars absorb toxic ions to a lesser magnitude as compared to salt susceptible ones. The uptake of ions in higher quantities results in burning symptoms of leaves and ultimately leading to plant death (MUNNS et al., 1983; FLOWERS and FLOWERS, 2005; AKRAM et al., 2009, 2010). In view of a number of reports it is now evident that soil pH, composition and concentration of elements, climatic conditions, soil textural class, quality of irrigation waters and type of plant species affect the nutrient dynamics and their absorption by plants under saline conditions (GRATTAN and GRIEVE, 1999; ZHU, 2003; MUNNS, 2005; AKRAM et al., 2010). For instance, high soil Na+ impairs the Ca2+ activity in the external medium, thereby restricting its availability in plants e.g., in Celosia argentea (CARTER et al., 2006), Setaria verticillata (HORST et al., 2006), and maize (SUAREZ and GRIEVE, 1988). It is well established that mineral nutrients acting synergistically or antagonistically may imbalance the nutrition of plants under saline environments. The defi ciencies of most of the nutrients commonly occur due to higher absorption of Na+ and Cl- by plant tissues (GRATTAN and GRIEVE, 1999; SUBBARAO et al., 2003; HU et al., 2005; ASHRAF et al., 2010). In view of these reports, it is likely that reduction in uptake of mineral nutrients under saline conditions may occur due to Na+-induced blockage or reduced activity of membrane ion transporters. Similarly, potassium uptake is perturbed by salinity thereby resulting in a reduced K+/Na+ ratio (IZZO et al., 1991; SUBBARAO et al., 1990). However, high K+/Na+ ratio in plants under saline conditions has been suggested as an important selection criterion for salt tolerance (SHAH et al., 1987; REYNOLDS et al., 2005). Plant hormones affect plant growth in multifarious ways affecting a number of physiological/biochemical processes in plants subjected to biotic and abiotic stresses (REYMOND and FARMER, 1998; STEDUTO et al., 2000; ASHRAF et al., 2008, 2010). Salicylic acid (SA) is one such plant growth regulators, which participate in the regulation of a number of physiological events taking place in the plant (DOLATABADIAN et al., 2008; ASHRAF et al., 2010). SA regulates some key plant functions such as stomatal functioning (ALDESUQUY et al., 1998), ion uptake and transport (GLASS, 1975; KAYDAN et al., 2007), photosynthesis (NOREEN and ASHRAF, 2008), water relations (BARKOSKY and EINHELLIG, 1993), production rate and content of anthocyanin and chlorophyll (KHURANA and MAHESHWARI, 1980). It also increases growth (ARFAN et al., 2007; ASHRAF et al., 2010), and up-regulates antioxidative system (NOREEN et al., 2009). All these functions have a signifi cant role in plant tolerance to salinity (NOREEN and ASHRAF, 2008; NOREEN et al., 2009; DOLATABADIAN et al., 2008; ABREU and MUNNE-BOSCH, 2009; ASHRAF et al., 2010). Furthermore, SA can greatly perturb the trans-membrane electrochemical potential of mitochondria and the ATP-dependent proton gradient of tonoplast-enriched vesicles (MACRI et al., 1986). In view of all the above reports, the principal objective of the present study was to appraise whether varying levels of exogenously applied SA could alleviate the adverse effects of salt stress on photosynthetic pigments, water relations and ion accumulation in sunfl ower plants. Furthermore, some key biochemical processes infl uenced by SA were identifi ed that could be used as selection criteria in sunfl ower under saline conditions. Materials and methods To assess the infl uence of foliar-applied salicylic acid to offset the salt-induced adverse effects on chlorophyll pigments, water relation components and accumulation of some key inorganic nutrients in sunfl ower (Helianthus annuus L.), an experiment was conducted at the Botanical Garden of the University of Agriculture, Faisalabad, Pakistan under the growth conditions as described earlier (NOREEN and ASHRAF, 2008). Two sunfl ower lines (Hysun-33 and SF-187) 170 S. Noreen, M. Ashraf, N.A. Akram Infl uence of exogenously applied salicylic acid on sunfl ower were subjected to two saline regimes i.e., 0 and 120 mM NaCl prepared in the modifi ed full strength Hoagland’s nutrient solution. Different concentrations (100, 200 and 300 mg L-1) of salicylic acid (C7H6O3) were prepared and pH of the solutions was adjusted at 5.5. Salicylic acid was applied exogenously in combination with 0.1 percent (v/v) tween-20 as a surfactant to ensure spreading of the applied solution on the leaf surface so as to attain maximal penetration into the leaf tissues. The plants were harvested 21 days after the foliar application of SA at the vegetative stage and data for following attributes were recorded: Photosynthetic pigments Chlorophylls a and b were appraised following ARNON (1949). The leaf samples (0.2 g each) were prepared by cutting the leaves into small pieces having 0.5 cm size. The extraction of the leaf samples was done in 80% acetone at -4 °C for 24 h. The extracted material was centrifuged at 10, 000 x g for 5 min. The absorbance of the supernatant was recorded by using a UV-Visible spectrophotometer (Model Hitachi-U 2001, Tokyo, Japan) at 645 and 663 nm against a blank containing 80% acetone. Leaf water potential (Ψw) A Scholander type water potential measuring system (Cook and Sons, Birmingham, England) was used to measure leaf water potential. A fully expanded youngest leaf from the top was cut from each plant and measurements were made following SCHOLANDER et al. (1965). Leaf osmotic potential (Ψs) The leaf osmotic potential was measured following WILSON et al. (1980). The leaf material was kept in polypropylene micro-centrifuge tubes containing capacity of 2 cm3. The tubes were frozen at -45 °C for one week. The material was thawed and homogenized with a tissue grinder. Then the homogenized material was centrifuged at 8000 x g for 4 min. A 10 µL was used in a Wescor-5500 vapor pressure osmometer for measuring osmotic potential. Leaf turgor potential (Ψp) The leaf turgor pressure was determined using the following equation: Ψp = Ψw – Ψs Relative water content (RWC) The fully expanded leaves were collected randomly from each plant from each replicate. After washing the samples were weighed and these values referred to as initial readings. Then, the leaf samples were placed in distilled water for 3 h in the dark at room temperature. The turgid leaves were blotted dry and weighed. After weighing, the material was oven-dried at 80 °C for 24 h. RWC of the leaves was appraised as described by JONES and TURNER (1980) using the following formula: RWC (%) = [(f. wt. – d. wt.)/(t. wt. – d. wt.)] x 100 where f.wt, d.wt, and t.wt are the fresh, oven dry, and turgid weights, respectively. Free proline content Free proline was determined following BATES et al. (1973). Leaf tissue (0.5 g) was ground with 10 mL of 3% (w/v) sulfo-salicylic acid solution. The sample fi ltrate containing 2.0 mL was reacted with 2.0 mL acid ninhydrin solution (1.25 g ninhydrin mixed with 30 mL glacial acetic acid), 20 mL (6M orthophosphoric acid) and 2.0 mL M orthophosphoric acid) and 2.0 mL M glacial acetic acid. The reaction was done at 100 °C, and terminated in an ice bath. A continuous stream of air was passed through the reaction mixture added with 4 mL toluene. The absorbance of the supernatant was read at 520 nm using a spectrophotometer (Model Hitachi U 2001 Tokyo, Japan). Proline concentration was worked out using the following formula: Free proline (µmol g-1 fresh weight) = Proline (µg ml-1) x Volume of toluene (mL)/(Mol. wt. of proline (g mol-1) x Leaf tissue fresh weight (g) Analysis of nutrients The oven-dried plant material (0.1 g) was ground and passed through a 40-mesh screen for chemical analysis. A digestion mixture consisting of H2SO4 and H2O2 was used to digest the material. The chemical analysis of different ions (K+, Ca2+, Na+) was carried out following WOLF (1982). Determination of Na+, K+ and Ca2+ Different concentrations of standards for sodium (Na+), potassium (K+) and calcium (Ca2+) were prepared. The standard curves of these ions were drawn using a fl ame photometer (Jenway-PFP7, ELE Instrument Co, Ltd. England). The samples were run for the determination of Na+, K+ and Ca2+ and the values of the ions in the unknown samples were worked out using the appropriate standard curves. Determination of chloride (Cl-) The chloride content was determined by extracting plant material (0.5 g) with 10 mL distilled de-ionized water. The material was heated at 80 °C till the volume was reduced to half. The volume of the solution was again made to 10 mL with distilled water. Chloride Analyzer (Model 926, Sherwood Scientifi c Ltd, Cambridge, UK) was used to determine the chloride content in the extracted samples. Statistical analysis of data Analysis of variance (ANOVA) of the data for all attributes was worked out using the COSTAT computer package (Cohort, Berkely, California). The least signifi cance difference test was employed following SNEDECOR and COCHRAN (1980) to assess whether mean values differed signifi cantly. Results Data presented in Tab. 1 for percent inhibition in shoot fresh and dry biomass showed that although salt stress caused a marked reduction in shoot biomass, foliar-applied salicylic acid reduced the salt- induced inhibition in these growth attributes in both hybrid lines of sunfl ower. Of all SA levels, 200 and 300 mg L-1 were relatively more effective in improving growth measured in terms of shoot biomass. Addition of salt (NaCl) to the root growing medium markedly reduced chlorophyll a in both hybrid lines. However, values of chlorophyll a were found similar in both hybrid lines (Tab. 2). The foliar spray of 200 or 300 mg L-1 SA caused a signifi cant improvement in chlorophyll a content in both sunfl ower hybrid lines grown under saline and non-saline regimes (Fig. 1). However, under salt treatment, foliar applied 300 mg L-1 SA signifi cantly improved chlorophyll a in line SF-187, while, 200 mg L-1 of SA in hybrid line Hysun-33 (Fig. 1). Salt stress signifi cantly reduced chlorophyll b in both hybrid lines (Tab. 2; Fig. 1). Line Hysun-33 maintained greater Infl uence of exogenously applied salicylic acid on sunfl ower 171 content of chlorophyll b than that in SF-187 under saline conditions. Salicylic acid improved chlorophyll b. Of all SA levels, 300 mg L-1 was found better than the others. The chlorophyll a/b ratio of the sunfl ower hybrid lines was not infl uenced by salinity. However, there were signifi cant differences between the hybrid lines in terms of chlorophyll a/b ratio. Leaf water potential (Ψw) of both sunfl ower lines was signifi cantly reduced due to salinity, and more marked being in line SF-187 (Tab. 2; Fig. 1). Exogenous application of varying levels of SA did not change the leaf water potential of sunfl ower hybrid lines under non-stressed conditions, whereas, under salinity stress, foliar application of all varying levels of SA caused a signifi cant decrease (more negative increase) in leaf water potential, particularly, in line Hysun-33 (Fig. 1). Imposition of salinity had a signifi cant reducing effect on leaf osmotic potential (Ψs) of both sunfl ower lines (Tab. 2; Fig. 1). Lines differed only under saline conditions. Line SF-187 had lower leaf osmotic potential than that in line Hysun-33 under salt-stressed conditions. Foliar spray of SA did not affect leaf osmotic potential of the hybrid lines under non-stress conditions, whereas under saline regimes, all SA levels caused a decrease in leaf osmotic potential but only in line SF-187. Leaf turgor potential (Ψp) remained almost unaltered due to imposition of salt stress (Tab. 2; Fig. 1). Both hybrid lines did not signifi cantly differ in leaf turgor pressure. Foliar-applied 200 or 300 mg L-1 SA resulted in enhanced leaf turgor potential of salt stressed plants of line SF-187, whereas the reverse was true in line Hysun-33. Relative water content (RWC) of sunfl ower plants was signifi cantly decreased because of raising salinity of the growth medium (P ≤ 0.05) (Tab. 2; Fig. 2). Moreover, both lines also differed signifi cantly in this parameter. Foliar-applied SA did not signifi cantly alter leaf RWC in the salt-stressed or non-stressed plants of both lines. Values for RWC did not vary signifi cantly due to exogenous application of SA under salt-treated and untreated plants. Proline content differed signifi cantly due to different salt and SA treatments. However, both sunfl ower lines did not differ signifi cantly in proline content. Moreover, there were non-signifi cant interactions between salinity, SA or hybrid lines. Proline content increased with increasing levels of SA under saline and non-saline conditions. Line Hysun-33 maintained higher content of proline compared to that in line SF-187. Both lines, Hysun-33 and SF-187, contained higher content of proline under salt stress than that under normal conditions (Tab. 2; Fig. 2). Concentrations of Na+ in the leaves and roots increased signifi cantly due to imposition of salt stress in the growth medium (Tab. 2). Both lines did not show difference in leaf Na+, whereas they differed signifi cantly in root Na+. Exogenous application of different levels of SA had a slight decreasing effect on Na+ content in the leaf tissues of salt stressed plants of both lines, particularly after foliar application of 300 mg L-1 SA (Fig. 2). On the other hand, foliar-applied SA at different concentrations produced inconsistent pattern of Na+ accumulation by the roots of sunfl ower hybrid lines (Fig. 2). The addition of NaCl to the growth medium resulted in a signifi cant reduction in root K+ content of the hybrid lines. Both sunfl ower lines also differed signifi cantly in this physiological attribute (P ≤ 0.05). The accumulation of K+ by the leaf or root tissues was not affected signifi cantly due to foliar spray of SA. However, a slight improvement in this ion content was observed in both hybrid lines under saline and non-saline regimes. The SA application resulted in a slight increase in the uptake of K+ by the root tissues in line Hysun-33 as compared to that in line SF-187 under NaCl treatment (Fig. 2). The concentration of Ca2+ in the leaves of Hysun-33 was higher than that in line SF-187 grown under non-saline conditions. Contrarily, the pattern of accumulation of Ca2+ in the leaves of both lines was inconsistent under salinity stress (Tab. 2; Fig. 3). However, accumulation of Ca2+ in the roots of line SF-187 was elevated as compared to that in line Hysun-33 under salt-stressed and non- stressed conditions. Foliar application of 100 mg L-1 SA resulted in increased uptake of Ca2+ by leaf tissues of line Hysun-33 under salt- stressed conditions. Furthermore, foliar spray of 200 and 300 mg L-1 SA resulted in a maximal increase in root Ca2+ in the sunfl ower hybrid lines under salt stress and non-stress substrates (Fig. 3). The plants grown under NaCl-treated regime showed a signifi cant effect on the uptake of Cl- by leaves and roots of the sunfl ower hybrid lines under salt-stressed and non-stress regimes (Tab. 2). There was no signifi cant difference in both lines in leaf or root Cl- content (Fig. 3). Furthermore, accumulation of Cl- by leaf or root tissues was little affected by foliar application of SA under saline or non-saline Tab. 1: Percent inhibition in shoot biomass due to salt stress (120 mM NaCl) and percent improvement due to foliar-applied varying levels of salicylic acid of two lines of sunfl ower. Salicylic acid (SA) Percent (%) inhibition under saline conditions Shoot fresh weight Shoot dry weight SF-187 Hysun-33 SF-187 Hysun-33 0 mg L-1 39.47 50 24.4 42.1 100 mg L-1 40.8 49.9 27.3 45.4 200 mg L-1 41.4 46.5 47.9 41.0 300 mg L-1 38.6 44.08 36.2 37.6 Percent (%) improvement due to foliar-applied SA under normal and saline conditions Shoot fresh weight Shoot dry weight SF-187 Hysun-33 SF-187 Hysun-33 Control Saline Control Saline Control Saline Control Saline 100 mg L-1 12 9.47 4.44 4.75 9.5 13.2 8.2 1.9 200 mg L-1 26.13 22.08 4.62 11.9 15.7 20.6 11.4 9.8 300 mg L-1 14.24 15.93 10.5 23.6 2.53 13.7 16.8 25.9 172 S. Noreen, M. Ashraf, N.A. Akram Infl uence of exogenously applied salicylic acid on sunfl ower Tab. 2: Analyses of variance of the data (Mean squares) for chlorophyll pigments, water relation components, free proline and concentrations of inorganic nutrients in two sunfl ower (Helianthus annuus L.) hybrid lines when varying levels of salicylic acid were applied as a foliar spray to 24-day old plants subjected to normal or saline conditions. Sources of variation df Chlorophyll a Chlorophyll b Chlorophyll Leaf water Leaf a/b ratio potential osmotic potential Salt (S) 1 3.21*** 0.82*** 0.284ns 2.024*** 2.043*** Hybrid lines (HBL) 1 0.095ns 0.027* 0.825* 0.007ns 0.157** Salicylic acid (SA) 3 0.246* 0.009ns 0.394ns 0.035*** 0.018ns S x HBL 1 0.054ns 0.006ns 0.002ns 0.073*** 0.288*** S x SA 3 0.021ns 0.009ns 0.065ns 0.007ns 0.023ns HBL x SA 3 0.06ns 0.003ns 0.087ns 0.064*** 0.018ns Salt x HBL x SA 3 0.084ns 0.002ns 0.148ns 0.005ns 0.003ns Error 48 0.068 0.005 0.184 0.005 0.016 Leaf turgor Relative water Leaf free Leaf Na+ Root Na+ potential content proline Salt (S) 1 0.0004ns 2635.4*** 51.12*** 2053.2*** 4856.3*** Hybrid lines (HBL) 1 0.099* 141.6** 0.004ns 3.376ns 481.3** Salicylic acid (SA) 3 0.039ns 26.0ns 2.416** 8.471ns 77.61ns S x HBL 1 0.071ns 1.28ns 1.062ns 0.083ns 549.3** S x SA 3 0.013ns 13.52ns 0.08ns 6.74ns 104.8ns HBL x SA 3 0.095* 29.43ns 0.064ns 5.83ns 47.41ns Salt x HBL x SA 3 0.01ns 16.18ns 0.527ns 1.365ns 25.84ns Error 48 0.023 14.91 0.561 3.757 63.42 Leaf K+ Root K+ Leaf Ca2+ Root Ca2+ Leaf Cl- Salt (S) 1 7255.8*** 4863.7*** 73.68*** 454.9*** 4728.3*** Hybrid lines (HBL) 1 24.47ns 111.2* 2.939ns 44.76** 7.548ns Salicylic acid (SA) 3 108.7* 28.22ns 2.925ns 65.48*** 13.52ns S x HBL 1 16.72ns 3.478ns 1.67ns 11.41ns 27.59ns S x SA 3 12.48ns 5.03ns 11.81*** 1.767ns 15.33ns HBL x SA 3 2.898ns 10.0ns 2.574ns 7.422ns 7.317ns Salt x HBL x SA 3 10.7ns 6.70ns 9.32** 3.299ns 1.453ns Error 48 30.05 23.68 1.462 5.13 24.96 Root Cl- Leaf Root Leaf Root K+/Na+/Na+ + K+/Na+/Na+ + Ca2+/Na2+/Na2+ + Ca2+/Na2+/Na2+ + Salt (S) 1 2499.0*** 782.4*** 57.87*** 23.87*** 7.49*** Hybrid lines (HBL) 1 2.074ns 1.64ns 6.54*** 0.226ns 2.02*** Salicylic acid (SA) 3 8.56ns 4.44ns 0.234ns 0.232ns 0.26** S x HBL 1 25.6ns 2.99ns 9.37*** 0.097ns 1.387*** S x SA 3 5.67ns 2.026ns 0.154ns 0.317ns 0.038ns HBL x SA 3 5.1ns 2.756ns 0.232ns 0.123ns 0.05ns Salt x HBL x SA 3 3.18ns 1.503ns 0.127ns 0.199ns 0.069ns Error 48 8.55 1.647 0.296 0.132 0.053 *, **, *** = signifi cant at 0.05, 0.01 and 0.001 levels. ns = non-signifi cant regimes. Salt stress had a signifi cant decreasing effect on leaf and root K+/Na+ ratios of both sunfl ower lines. In contrast, under saline conditions, at 200 mg L-1 of SA a slight increase in K+/Na+ ratio was observed in cv. SF-187. Foliar-applied varying levels of salicylic acid showed inconsistent results for these ionic ratios (Tab. 2; Fig. 4). Leaf and root Ca2+/Na+ ratios were signifi cantly (P ≤ 0.001) in- fl uenced by salt stress. A non-signifi cant effect of exogenously applied salicylic acid was observed on leaf Ca2+/Na+ ratio, while root Ca2+/Na+ was increased due to SA application. Of all SA levels, 300 mg L-1 was highly effective for improving tissue Ca2+/Na+ ratios in both sunfl ower hybrids. Of both sunfl ower hybrids, SF-187 was better than Hysun-33 in this attribute (Tab. 2; Fig. 4). Discussion Salicylic acid (SA) being a vital plant growth regulator as well as an antioxidant (RASKIN et al., 1992) has a potential to allay the salt- Infl uence of exogenously applied salicylic acid on sunfl ower 173 Fig. 1: Chlorophyll a and b contents, chlorophyll a / b ratio, and leaf water, osmotic and turgor potentials of two sunfl ower (Helianthus annuus L.) hybrid lines when varying levels of salicylic acid were applied as a foliar spray to 24 day-old plants subjected to normal or saline conditions (Mean ± S.E.; n = 4). Fig. 2: Relative water content, proline accumulation, and leaf and root Na+ and K+ contents of two sunfl ower (Helianthus annuus L.) hybrid lines when varying levels of salicylic acid were applied as a foliar spray to 24 day-old plants subjected to normal or saline conditions (Mean ± S.E.; n = 4). 174 S. Noreen, M. Ashraf, N.A. Akram Infl uence of exogenously applied salicylic acid on sunfl ower induced harmful effects on crop growth and development (EL-TAYEB, 2005; ARFAN et al., 2007; ASHRAF et al., 2010). Photosynthetic pigments such as chlorophyll a and b are chief components of photosystems driving the mechanism of photosynthesis and hence growth in terms of biomass production or seed yield. In the present study, the contents of photosynthetic pigments was slightly improved with SA application. These fi ndings are similar to those of GHAI et al. (2002) who showed a considerable improvement in chlorophyll contents due to foliar applied SA (200 mg L-1). Similarly, FARIDUDDIN et al. (2003) reported that Brassica juncea plants sprayed with Fig. 3: Leaf and root Ca2+ and Cl- accumulation in two sunfl ower (Helianthus annuus L.) hybrid lines when varying levels of salicylic acid were applied as a foliar spray to 24 day-old plants subjected to normal or saline conditions (Mean ± S.E.; n = 4). Fig. 4: Leaf and root K+/ Na+ and Ca2+/ Na+ ratios of two sunfl ower (Helianthus annuus L.) hybrid lines when varying levels of salicylic acid were applied as a foliar spray to 24 day-old plants subjected to normal or saline conditions (Mean ± S.E.; n = 4). Infl uence of exogenously applied salicylic acid on sunfl ower 175 10-5 M of SA showed 20 percent higher chlorophyll than those sprayed with water only. By contrast, no change in chlorophyll content was observed in corn and soybean plants exogenously supplied with acetyl salicylic acid (KHAN et al., 2003). In the present study, a positive correlation of photosynthetic effi ciency (Astudy, a positive correlation of photosynthetic effi ciency (Astudy, a positive correlation of photosynthetic effi ciency ( ) with chlorophyll a or chlorophyll b (r = 0.416**; 0.436**, respectively) r = 0.416**; 0.436**, respectively) r has been observed. Such relationships between A and chlorophyll contents have earlier been reported by FARIDUDDIN et al. (2003). They found that exogenous SA application caused an improvement in photosynthetic capacity in salt stressed Brassica juncea plants in association with improved chlorophyll content. The reduction in chlorophyll contents may occur due to acceleration in chlorophyll degradation or reduction in chlorophyll synthesis. However, it has been reported that imposition of salt stress causes deterioration in the structure of chloroplast e.g., thylakoid membranes and plastids due to direct Na+ toxicity and cellular oxidative damage (MITTLER, 2002). In the present study, a strong negative correlation between leaf Na+ and each of leaf chlorophyll a, chlorophyll b, or A (r = -0.610***; r = -0.610***; r -0.804*** and -0.527*** respectively) has been observed. Maintenance of plant water status or osmoregulation is a vital physiological process for maintaining optimal plant growth (TAIZ and ZEIGER, 2006). In the present study, leaf water relation parameters, leaf turgor, water and osmotic potentials, and relative water content of both sunfl ower lines were adversely affected by salt stress. Application of 200 or 300 mg L-1 SA improved leaf relative water content (RWC), but decreased leaf osmotic potential and leaf Ψw. Moreover, leaf RWC and leaf Ψp of the salt stressed plants of line SF-187 were higher than those in the leaves of line Hysun-33. Leaf osmotic potential of the salt stressed plants of SF- 187 was also reduced due to SA application. These fi ndings are in agreement with an earlier study (ASHRAF, 1989) in which leaf turgor potential increased with a concomitant decrease in osmotic potential in blackgram (Vigna mungo) under salt stress. Decrease in osmotic potential may occur due to accumulation of inorganic and/or organic solutes. Of various organic solutes, glycinebetaine, amino acids mainly proline, and soluble sugars play important roles in osmotic adjustment (HASEGAWA et al., 2000; ASHRAF and FOOLAD, 2005, 2007). Among inorganic solutes, K+ and Na+ are considered very important because they also play a vital role in osmoregulation, but Na+ is potentially damaging when compared either with K+ or organic solutes (SUBBARAO et al., 2001; TESTER and DAVENPORT, 2003). However, exogenous application of SA decreased leaf Na+ content in the salt stressed plants of both sunfl ower lines, but leaf K+ remained almost unaffected in both shoots and roots of both lines due to foliar-applied SA application. If relationships are worked out between leaf Ψs and each of proline, leaf Na+, or leaf K+ (leaf OP vs leaf proline; leaf OP vs leaf Na+; leaf OP vs leaf K+, r = 0.625***; 0.652***; -0.649***), it is amply clear that leaf r = 0.625***; 0.652***; -0.649***), it is amply clear that leaf r Na+ and leaf proline had a marked contribution in osmoregulation. Increased accumulation of proline in the salt stressed plants of both sunfl ower lines is similar to the fi ndings of SHAKIROVA et al. (2003) who reported that SA-treated wheat seedlings showed enhanced accumulation of free proline. This can be further supported by the inference drawn by ASHRAF and HARRIS (2004) that under salt stress, higher accumulation of proline is one of the vital strategies of plants for causing osmotic adjustment, particularly in the presence of high cytosolic Na+. In view of the results from the present study and all these reports, it can be suggested that although exogenous SA reduced accumulation of Na+ in the leaves, its concentration is high enough to cause toxic effects, so increased accumulation of proline in the salt stressed sunfl ower plants reduced the toxic effect of Na+ during osmotic adjustment. The results for accumulation of different mineral ions in the root and shoot tissues of both sunfl ower lines showed enhanced accumulation of Na+ and Cl- accompanied with a decline in K+ and Ca2+ in both sunfl ower lines under saline substrate. These results support the viewpoint that plants subjected to saline substrate are prone to specifi c ion toxicity, ionic imbalance, and nutrient defi ciency (ASHRAF, 1994, 2004). However, exogenously applied SA reduced leaf Na+ of both sunfl ower lines. These results support the earlier fi ndings of EL-TAYEB (2005) in which SA application resulted in reduced Na+ in the leaves of barley seedlings under salt stress. In contrast, K+ accumulation remained almost unchanged due to SA application. However, root or leaf Ca2+ content of salt stressed plants of both sunfl ower lines increased due to exogenously applied SA. These results support the earlier fi ndings of KAWANO and MUTO (2000) who observed that SA enhanced the cytosolic Ca2+ level in tobacco cell suspension culture. In view of these results and some published reports available in the literature, it is suggested that exogenous SA application might have elevated cytosolic Ca2+ that acted as a second messenger taking part in a multitude of physiological responses including expression of osmotic responsive genes (PARDO et al., 1998) and antioxidant enzymes (CHEN and LI, 2001; AGARWAL et al., 2005). To appraise salt tolerance, K+/Na+ and Ca2+/Na+ ratios are considered as potential selection criteria, however, in the present study salt stress signifi cantly decreased the leaf and root K+/Na+ ratios, while Ca2+/ Na+ ratios were not infl uenced in sunfl ower plants and foliar-applied salicylic acid showed inconsistent results for these ionic ratios. In conclusion, salt stress adversely affected the growth, chlorophyll pigments, water relations and contents of some key mineral nutrients, while increased the amount of proline and leaf and root Na+ as well as Cl- contents in both sunfl ower lines. Of both sunfl ower lines, Hysun-33 had higher amounts of photosynthetic pigments and essential nutrients than did SF-187. Foliar-applied SA improved growth, chlorophyll a and b pigments, leaf turgor potential, and leaf and root Ca2+ concentrations. 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