Changes in the Levels of Jasmonates and Free Polyamines Induced by Na2so4 and Nacl in Roots and Leaves of the Halophyte Prosopis Strombulifera

Biologia 67/4: 1—, 2012 Section Botany DOI: 10.2478/s11756-012-0052-7

Changes in the levels of jasmonates and free polyamines induced by Na2SO4 and NaCl in roots and leaves of the halophyte Prosopis strombulifera

Mariana A. Reginato1, Guillermina I. Abdala1, Otto Miersch2,OscarA.Ruiz3, Elsa Moschetti5 &VirginiaLuna1*

1Dpto. Ciencias Naturales, Universidad Nacional de Río Cuarto, 5800–Río Cuarto, Córdoba, Argentina; e-mail: [email protected] 2Leibniz-Institute of Plant Biochemistry, Department of Natural Product Biotechnology, Weinberg 3,D-06120 Halle (Saale), Germany 3Unidad de Biotecnología 1, IIB-INTECH/UNSAM-CONICET, 7130 Chascomús, Buenos Aires, Argentina; e-mail: [email protected] 4Dpto. de Matemática, Universidad Nacional de Río Cuarto, 5800–Río Cuarto, Córdoba, Argentina; e-mail: [email protected]

Abstract: Prosopis strombulifera, a common legume in high-salinity soils of Argentina, is a useful model for elucidation of salt tolerance mechanisms and specific biochemical pathways in halophytes, since its NaCl tolerance exceeds the limit described for most halophytic plants. We analyzed the effects of the increasing concentration of two main soil salts, Na2SO4 and NaCl, on growth parameters of P. strombulifera, chlorophyll levels, and content of jasmonates (JAs) and polyamines (PAs), which are key molecules involved in stress responses. P. strombulifera showed a halophytic response (growth promo- tion) to NaCl, but strong growth inhibition by iso-osmotic solutions of Na2SO4. Chlorophyll levels, number of leaves and 2− leaf area were also differentially affected. An important finding was the partial alleviation of SO4 toxicity by treatment with two-salt mixture. JAs are not directly involved in salt tolerance in this species since its levels decrease under all salt treatments. Beneficial effects of Putrescine (Put) accumulation in NaCl treated plants maybe inferred probably associated with the antioxidative defense system. Another novel finding is the accumulation of the uncommon PA cadaverine in roots 2− under high Na2SO4, which may be related to SO4 toxicity.

Key words: Prosopis strombulifera; halophytes; jasmonates; polyamines; NaCl; Na2SO4

Introduction tion than Cl−-based solutions, at iso-osmotic concentrations (Sosa et al. 2005; Llanes et al. 2005). P. strom- The genus Prosopis (Fabaceae subfamily Mimosoideae) bulifera seedlings grown in the presence of high NaCl is found in arid and semiarid regions worldwide, and concentrations do not develop salt glands in the leaves. is the major component of many native ecosystems in Some tissues display vacuolization, and the root system South and North America. Species of this genus of- undergoes precocious lignification and/or suberization ten have economic and ecological importance; e.g.,as of endodermal cells, with Casparian strips found much sources of shade, firewood, food, and forage for wildlife closer to the root tip than in glycophytes. These plants and livestock. Certain species, particularly P. pallida, can therefore more efficiently filter soil solution to pre- P. juliflora, P. tamarugo,andP. alba, display rapid vent passage of excess ions to the xylem (Reinoso et al. −1 growth at high (seawater level) salinity, ∼45 dS m , 2004). Na2SO4 treatment of P. strombulifera seedlings which is nearly 20-fold higher than salinity levels toler- induced structural alterations in cells and tissues, with able to annual temperate legumes (Felker 2007). consequent changes in growth patterns at various levels P. strombulifera (Lam.) Benth is a spiny shrub of organization, and anatomical and histological differ- common in high-salinity soils in Córdoba and San ences in roots, stems, and leaflets, compared to control Luis provinces, Argentina. Comparative studies have plants, or plants grown in high NaCl salinity (Reinoso 2− shown that SO4 -based solutions have considerably et al. 2005). Thus, P. strombulifera provides a useful stronger inhibitory effect on P. strombulifera germina- model for study of salt tolerance mechanisms, and spe-

* Corresponding author

c 2012 Institute of Botany, Slovak Academy of Sciences 2 M.A. Reginato et al. cific underlying biochemical pathways, in this genus and Material and methods halophytes in general. Plant materials and growth conditions Reactions to high salinity have been previously Seeds of P. strombulifera were collected in southwestern San studied in halophytic plants such as Thellungiella Luis province, Argentina. Pods were collected at random halophila (Gong et al. 2005), and in glycophytic plants from 100 plants within the same population, and peeled. such as Arabidopsis (Bressan et al. 2001). Thellungiella, Seeds were scarified with 98% sulfuric acid for 10 min, in comparison to Arabidopsis, maintains higher content washed overnight under running water, rinsed in distilled water, and germinated by placing in a Petri dish with two of various metabolites (fructose, sucrose, complex sug- ◦ ars, malate, proline) in the absence or presence of salt layers of water-saturated filter paper at 37 C for 24 h. stress. Many of the responses of Arabidopsis to high Germinated seedlings with 20mm-long roots were grown under hydroponic conditions in black 28 × 22 × 10 cm salinity can be viewed as extreme defensive reactions, trays (200 seedlings per tray) with Hoagland’s solution which expend energy on multiple metabolic pathways (10% of full-strength). The seedlings were self-supported at low stress levels. Thellungiella appears more efficient in small holes on the tray cover. The trays were placed in maintenance of energy and metabolite composition in a growth chamber (Conviron E15, Controlled Environ- (Gong et al. 2005). ments Ltd., Manitoba, Canada) under a cycle of 16 h light −2 −1 ◦ ◦ Extensive studies have been performed on growth (200 µmol m s )(28C): 8 h dark (20 C),70% relative responses, osmolytes, metabolites, ion accumulation, humidity. After one week, the nutrient solution was changed − etc. in salt tolerant plants, but information on related to 25% Hoagland’s (osmotic potential (Ψo) = 0.11 MPa). plant growth regulators is still limited. All media were maintained at pH 6. An aquarium tubing system with peristaltic pump was used for aeration. Each Responses of plants to biotic and abiotic stresses experiment was performed four times, consecutively (2 trays often involve generation of jasmonates (JAs) and per treatment each time). polyamines (PAs). JAs are key signaling molecules involved in diverse developmental processes and in plant Salt treatment stress responses (for review see Wasternack 2007). They When seedlings were 21day-old, salt treatments were started −1 −1 include jasmonic acid (JA), its methyl ester (JAME), by adding NaCl (50 mmol L )orNa2SO4 (38 mmol L ) amino acid conjugates, and metabolites such as 12-OH- pulses separately for single-salt treatments, or as iso-osmotic JA and 11-OH-JA. The octadecanoid cis (+) 12 oxo- mixture of the two salts (“bisaline treatment”), every 48 h − − phytodienoic acid (OPDA) is the precursor of JA. until reaching final osmotic potentials (Ψo) = 1.0, 1.9, or −2.6 MPa (measured by a vapor pressure osmometer PAs are involved in a wide variety of physiolog- Model 5500, Wescor Inc., Logan, UT, USA). These Ψo val- ical processes. Free PAs are small organic cations es- ues corresponded to salt concentrations of 250, 500 and sential for eukaryotic cell growth, and have been pro- 700 mmol L−1 NaCl and 189.7, 379.2 and 530.8 mmol L−1 posed as a new category of plant growth regulators Na2SO4 respectively. Plant age was 29, 40, and 48 d, re- (Liu et al. 2007). The three main PAs in plants are pu- spectively. Control plants remained in 25% Hoagland’s (Ψo trescine (Put), spermidine (Spd), and spermine (Spm). = −0.11 MPa). At each time point, roots and leaves of 30 Less common PAs are diaminopropane (Dap) and ca- control plants and 30 salt-treated plants were collected at daverine (Cad). random from each tray, frozen with liquid N2, and stored at − ◦ Accumulation of PAs under salt stress was re- 80 C for analysis of chlorophylls, JAs, and PAs. ported for mono- and dicotyledonous plants. Lupinus Growth parameters luteus seedlings accumulate Put and Spd in leaves in Leaf number, root length, and shoot height were measured response to increased NaCl (Legocka & Kluk 2005). at Ψo value −0.65 (day 25), −1.0 (day 29), −1.9 (day 40), Salt-tolerant cultivars of rice and tomato accumulated and −2.6 MPa (day 48) for 30 plants from each treatment. Spd and Spm, whereas salt-sensitive cultivars accumu- Total leaf area was determined from all the leaves of three th lated Put (Krishnamurthy et al. 1989; Santa Cruz et plants. Individual leaf area was determined from the 4 and th al. 1999). Many protective roles have been proposed for 5 leaves of the same three plants. Digital leaf images were PAs, e.g., scavenging free radicals, cellular pH mainte- created using a Hewlett Packard flat scanner, and analyzed nance, cellular ionic balance and membrane stabiliza- by the Image-Pro Plus 2.0 program. tion regulation of cationic channels, prevention of lipid Chlorophyll content peroxidation, and bioenergetics of photosynthesis. Such Levels of chlorophyll a and b were quantified by the con- processes may occur separately, or be combined in a ventional method, using the corresponding extinction coef- unified strategy to minimize membrane damage, pro- ficients for calculations. Fresh leaves were ground in a mor- mote cell growth, and enhance cell survival in response tar and left in 80% acetone 1 h at 4 ◦C for extraction. After to stress (Liu et al. 2007). centrifugation, absorbance of the supernatant was measured Using P. strombulifera as a model, we determined: at 650 nm (for chlorophyll a) and 665 nm (for chlorophyll b) using a spectrophotometer (Helios Gamma, Thermospec- (i) effects of increasing concentrations of Na2SO4,NaCl, and their iso-osmotic mixture on growth parameters tronic, UK). (shoot and root growth, leaf area, number of leaves and Extraction, purification, and estimation of jasmonates chlorophyll content); (ii) levels of endogenous JAs and JAs were estimated by the method of Miersch et al. (2008). 2 free PAs in plants grown under hydroponic conditions, 500 mg DW was homogenized and 100 ng ( H6)JA, 100 2 2 in order to clarify the role of these compounds in salt ng ( H5)OPDA, 100 ng 11-( H3)OAc-JA, or 100 ng 12- 2 tolerance. ( H3)OAc-JA added as internal standards. The homogenate Levels of jasmonates and free polyamines induced by Na2SO4 and NaCl in Prosopis strombulifera 3 was filtered under vacuum and the eluate was evaporated, Statistical analysis and the acetylation of endogenous hydroxylated JA was A simple randomized design with four treatments was used. ◦ performed with pyridine and acetic acid (2:1) at 20 C Two factors were considered for all variables: osmotic po- overnight. The extract was dried, dissolved in methanol, tential (−1.0, −1.9, or −2.6 MPa) and salt treatment (con- and loaded on columns containing 3 mL DEAE-Sephadex trol, NaCl, Na2SO4, or mixture of the two salts). An initial A25 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) descriptive analysis was made in each case, and examined − (Ac -form, methanol). The column was washed with 3 mL for normality (Shapiro-Wilk test) and variance homogene- methanol, then with 3 mL 0.1 M acetic acid in methanol, ity (Levenne test). In cases for which these parameters were and all eluates combined (neutral JAs). Further, elutes with verified, comparisons were performed by Student’s t-test, 3 mL 1 M acetic acid in methanol and 3 mL 1.5 M acetic or non-parametric Mann Whitney-U test, as appropriate. acid in methanol were collected and evaporated (acidic JAs). When significant differences were observed, the Tukey a Separation was performed on preparative HPLC with a Eu- posteriori test was applied. For JAs, comparisons were per- 2 rospher 100-C18 column (5 µm, 250 · 4mm), Solvent A: formed by non-parametric Kruskal-Wallis test; when signif- methanol, Solvent B: 0.2% acetic acid in H2O. Gradient 40– icant differences were observed, data were compared using −1 100% A in 25 min. Flow: 1 mL min .FractionsatRt 10– a non-parametric test. For PAs, ANOVA and a posteriori 11.30 min, 12–13.30 min, and 20.30–22 min were collected. Bonferroni test were used. P values < 0.05 were considered For derivatization, samples were dissolved in CHCl3/ statistically significant. N,N-diisopropylethylamine (1:1) and derivatized with pen- tafluorobenzylbromide at 20 ◦C overnight. Evaporated, dissolved in 7 mL n-hexane and passed through a SiOH-column Results (500 mg; Machery-Nagel, Duren, Germany). Pentafluo- robenzyl esters were eluted with 7 mL n-hexane/diethyl- Growth parameters ether (2:1), evaporated, dissolved in 100 µLMeCN.The NaCl treatment caused significant shoot growth stim- analysis was performed by GC-MS (Thermo Finnigan, San ulation at the start of salinization (−0.65 MPa, day Jose, CA, USA), 100 eV, negative chemical ionization. Injec- ◦ ◦ 25), and at low salt concentration (−1.0 MPa, day 29). tion temperature 250 C, interface temperature 275 C; he- − lium40cms−1; splitless injection. Only high NaCl salt stress ( 2.6 MPa) led to decrease The column temperature program was about 1 min in shoot length. On the other hand Na2SO4 treatment 60 ◦C, 25 ◦Cmin−1 to 180 ◦C, 5 ◦Cmin−1 to 270 ◦C, 10 ◦C caused strong shoot and root growth inhibition already −1 ◦ ◦ min to 300 C, 10 min 300 C; Rt of pentafluorobenzyl es- at moderate salinity (−1.9 MPa, day 40). This inhi- 2 2 ters: ( H6)JA 10.30 min, JA 10.36 min, 11-( H3)OAc-JA bition became more pronounced as salt concentration 2 13.60 min, 11-OAc-JA 13.63 min, 12-( H3)OAc-JA 15.39 2 increased, with visible chlorosis and toxicity symptoms min, 12-OAc-JA 15.42 min ( H5)OPDA 20.10 min, OPDA (−2.6 MPa, day 48). When NaCl and Na2SO4 were 20.16 min. Fragments m/z 209, 215 (standard), m/z 267, combined in iso-osmotic two-salt solution, the plant re- 270 (standard), m/z 267, 270 (standard), and m/z 291, sponse was intermediate between those observed with 296 (standard) were used for quantification of JA, 11- hydroxyjasmonate (11-OH-JA), 12-hydroxyjasmonate (12- each one-salt solution (25% growth inhibition at the OH-JA), and OPDA, respectively. These determinations end of the experiment) (Fig. 1A). were performed in Dr. Otto Miersch Lab, Leibniz-Institute Leaf number followed the same tendency that of Plant Biochemistry, University of Halle, Germany. shoot growth, with a pronounced inhibition from the beginning of the experiment in Na2SO4-treated plants, Extraction and estimation of free polyamines reaching 65% inhibition at the highest concentration Free PAs (Put, Spd, Spm, Dap, Cad) were estimated by − the method of Marcé et al. (1995) as dansyl-derivatives by (Ψo = 2.6). Visible chlorosis and toxicity symptoms reversed-phase HPLC for roots and leaves of control and finally caused leaf abscission in these plants. Applica- salt-stressed plants. 300 mg fresh weight (FW) of plant ma- tion of bisaline solution partially alleviated this effect, terial was extracted with 1 mL of 5% (v/v) perchloric acid with a final reduction similar to that caused by NaCl (PCA), and incubated overnight at 4 ◦C. Extracts were re- alone (20%) (Fig. 1B). covered by centrifugation, and 200 µL supernatant was dan- Root growth in NaCl-treated plants showed signifi- sylated in a mixture containing 100 µL saturated Na2CO3, cant stimulation from −1.0 MPa to −1.9 MPa, and then 200 µL dansyl chloride (5 mg mL−1 acetone), and 5 µLof declined. In Na2SO4-treated plants, root growth showed 100 mM 1,7-diaminoheptane as internal standard. The mix- significant inhibition from −1.9 MPa until the end of ture was incubated overnight in darkness at room temperature. The reaction was stopped by addition of 100 µL proline the experiment. With two-salt mixture, root growth −1 − (100 mg mL ), and dansylated PAs were extracted with showed stimulation at 0.65 MPa, and inhibition at 400 µL toluene. Organic phase was vacuum-evaporated, and higher Ψo values (Fig. 1C). dansylated PAs were dissolved in 100 µL acetonitrile. Total leaf area was lower in Na2SO4-treated than Polyamine content was analyzed by HPLC (ISCO 2350, in control plants at Ψo = −1.9, and the reduction coupled to a fluorescent detector Varian Fluorichom) with reached 60% at Ψo = −2.6 (Fig. 2A). Individual leaf a Sephasil C-18 reverse phase column (Amersham Pharma- area was affected only by Na2SO4 treatment; it was cia). Mobile phase consisted on water:acetronitrile in a pro- reduced 30% relative to controls at Ψo = −2.6 MPa gramed gradient (0–4.5 min. 30% water:70% acetonitrile; (Fig. 2B). Levels of chlorophyll a and b were af- 4.5–9.5 min. 100% acetonitrile; 9.5–22 min. 30% water: 70% acetronitrile) at a flow rate of 1.5 mL min−1.Bycompari- fected differently by one-salt and two-salt treatments. son of the peak areas of polyamine standards (Sigma) at the Chlorophyll a decreased ∼15% relative to control under corresponding retention time (Kobats 1958), the amount of Na2SO4 treatment (Fig. 2C), with visible chlorosis of free PAs was calculated. leaves. Under NaCl or two-salt treatment, chlorophyll b 4 M.A. Reginato et al.

Fig. 2. Effects of NaCl, Na2SO4, and their two-salt mixture on total leaf area (A) at low (−1.0 MPa, day 29), moderate (−1.9 MPa, day 40), and high salinity (−2.6 MPa, day 48), and individual leaf Fig. 1. Effects of NaCl, Na2SO4, and their two-salt iso-osmotic area (B) and chlorophyll content (C) of P. strombulifera plants mixture on growth of P. strombulifera plants at the begin- at high salinity (Ψo = −2.6 MPa, day 48). Columns represent av- ning of salt treatment (−0.65 MPa, day 25), and under low erage values from four independent experiments, with standard (−1.0 MPa, day 29), moderate (−1.9 MPa, day 40), and high error (S.E.) shown by bars. *, statistically significant difference salinity (−2.6 MPa, day 48). (A) Relative shoot growth. (B) (P <0.05). Picture: fifth leaves of control and salt-treated plants. Number of leaves (C) Relative root growth. The horizontal line at 100% represents growth of control plants. Values are means from four replicate experiments. *, statistically significant difference (P <0.05). Changes in content of JAs under salt stress OPDA (JA precursor), JA itself, and JA derivatives (11-OH-JA and 12-OH-JA) were all detected in P. decreased, but symptoms of chlorosis were not evident strombulifera seedlings. (Fig. 2C). No changes in individual leaf area or chloro- Levels of JAs in leaves and roots of control and phyll a content were observed at −1.0 or −1.9 MPa salt-treated plants are summarized in Table 1. OPDA (data not shown). was the major component in roots and leaves of con- Levels of jasmonates and free polyamines induced by Na2SO4 and NaCl in Prosopis strombulifera 5

Table 1. Eﬀects of NaCl, Na2SO4 and their iso-osmotic mixture on JAs levels in leaves and roots of P. strombulifera plants at −1MPa, 29 d; −1.9MPa,40d;and−2.6 MPa, 48 d.

Days of Osmotic potential Organ Treatment OPDA JA 12-OH-JA 11-OH-JA culture (MPa) (pmol g−1 DW) (pmol g−1 DW) (pmol g−1 DW) (pmol −1 DW)

29 days –0.11 Leaves Control 4232.0 ± 949 1818.4 ± 899 3936.7 ± 1671 2045.5 ± 118 –1 Na2SO4 3889.5 ± 528 2818.5 ± 1718 2750.7 ± 817 2165.6 ± 569 –1 NaCl 3979.2 ± 199 1481.5 ± 897 3355.5 ± 1030 1706.5 ± 271 –1 NaCl + Na2SO4 4040.7 ± 113 1981.7 ± 830 2993.5 ± 991 2092.7 ± 1094 –0.11 Roots Control 17722.3 ± 4780a 3609.7 ± 2016 1123.0 ± 433 839.5 ± 284 a –1 Na2SO4 15621.3 ± 4567 1283.5 ± 1066 676.4 ± 320 569.8 ± 279 –1 NaCl 2582.6 ± 270b 1673.2 ± 1142 902.6 ± 250 441.0 ± 211 c –1 NaCl + Na2SO4 8125.2 ± 2236 4602.0 ± 1718 1105.8 ± 260 672.8 ± 186

40 days –0.11 Leaves Control 4131.3 ± 134a 2375.0 ± 732 2977.2 ± 1069 1367 ± 440 b –1.88 Na2SO4 2583.0 ± 590 2779.0 ± 1489 2333.5 ± 1312 2250.6 ± 133 –1.88 NaCl 1937.5 ± 51b 1943.0 ± 802 4028.0 ± 1871 2043.7 ± 695 b –1.88 NaCl + Na2SO4 2475.2 ± 86 1785.0 ± 336 2314.6 ± 770 1506.3 ± 192 –0.11 Roots Control 5955.2 ± 2736a 3056.8 ± 1100 930.2 ± 407 771.2 ± 376 a –1.88 Na2SO4 7855.0 ± 684 2162.7 ± 573 981.6 ± 332 496.8 ± 256 –1.88 NaCl 2620.0 ± 875b 1472.5 ± 569 765.7 ± 608 593.0 ± 231 b –1.88 NaCl + Na2SO4 2360.2 ± 752 1477.2 ± 436 1257.4 ± 509 512.8 ± 126

48 days –0.11 Leaves Control 3903.0 ± 110a 2400.2 ± 1243a 4196.7 ± 2076 1506.6 ± 1057 b b –2.6 Na2SO4 2084.6 ± 93 1297.6 ± 227 1516.3 ± 273 986.7 ± 728 –2.6 NaCl 2046.7 ± 79b 1423.2 ± 617b 3526.2 ± 2190 1076.5 ± 235 b b –2.6 NaCl + Na2SO4 2381.2 ± 30 1307.4 ± 279 3977.0 ± 983 1857.3 ± 597 –0.11 Roots Control 7102.0 ± 1851a 3603.0 ± 1671 971.7 ± 505 709.2 ± 402 b –2.6 Na2SO4 1910.0 ± 663 1111.0 ± 763 945.4 ± 244 501.2 ± 230 –2.6 NaCl 1807.2 ± 497b 1478.4 ± 372 1561.6 ± 341 714.7 ± 278 b –2.6 NaCl + Na2SO4 2254.5 ± 559 2577.3 ± 1577 771.0 ± 217 416.7 ± 91

Values show the concentration of OPDA, JA, 12-OH-JA and 11-OH-JA. The data are the mean ± SD (n = 4). Means within a column followed by different letters indicate statistically significant differences (P < 0.05). trol plants, and its level was the most sensitive to salt stress. OPDA content was highest in roots of 29 day-old control plants. It decreased significantly under low or moderate NaCl treatment (−1.0 or −1.9 MPa). At high water potential (−2.6 MPa; day 48), OPDA decreased regardless of salt ionic composition. In the leaves OPDA content was not affected by low salinity (−1.0 MPa; day 29), but was reduced significantly by moderate (−1.9 MPa; day 40) or high (−2.6 MPa; day 48) salinity. JA content in roots did not differ significantly among low, moderate, and high salt treatments. JA content in leaves was significantly decreased at high salinity, but not at low or moderate salinity. 12-OH-JA and 11-OH-JA levels were higher in leaves than in roots, and were not affected by salt treat- Fig. 3. Distribution pattern of JAs in leaves and roots of control ment, regardless of osmotic potential or type of salt. and salt-treated P. strombulifera plants. Control plants (day 48); Content of JAs in leaves and roots of control and Na2SO4, NaCl, and two-salt treated plants (Ψo = −2.6 MPa, salt-treated plants at −2.6 MPa (day 48) is illustrated day 48). Area of circle corresponds to total content of JAs in leaves (top) and roots (bottom). Area of circle in leaves of control by pie charts in Fig. 3, for better understanding of dis- plants corresponds to 12005 pmol/g DW (100%). Areas of circles tribution patterns. The results indicate that the content (and portions thereof) for other plant organs and treatments are of total JAs was reduced by salt treatment, particularly proportional. Na2SO4.

Changes in content of free PAs under salt stress leaves its content decreased at −1.0 and −1.9 MPa. Content of PAs (Put, Spd, Spm, Dap) varied greatly Spd in roots showed a signiﬁcant increase in depending on the salt treatment and the organ (leaf vs. Na2SO4-treated plants at Ψo = −1.0 MPa, but root) (Table 2). In most cases, Put was the major PA decreased in NaCl- and two-salt treated plants at component. In Na2SO4-treated roots, Put content was this salinity. Spd in leaves decreased under NaCl signiﬁcantly higher at −1.0 and −2.6 MPa, while in the and Na2SO4 treatments at moderate salinity (Ψo = 6 M.A. Reginato et al.

Table 2. Eﬀects of NaCl, Na2SO4 and their iso-osmotic mixture on free PAs levels in leaves and roots of P. strombulifera plants at −1 MPa, day 29; −1.88 MPa, day 40; and −2.6 MPa, day 48.

Days of Osmotic potential Organ Treatment Put Spd Spm 1,3 Dap culture (MPa) (nmol g−1 FW) (nmol g−1 FW) (nmol g−1 FW)(nmol g−1 FW)

29 days –0.11 Leaves Control 470.1 ± 113a 62.0 ± 19 34.6 ± 6 284.2 ± 2.1 b –1 Na2SO4 84.5 ± 59 46.5 ± 17 34.0 ± 4.2 383.1 ± 70 –1 NaCl 714.9 ± 133a 45.6 ± 13 46.6 ± 11 354.9 ± 102 c –1 NaCl + Na2SO4 203.3 ± 153 40.6 ± 4.5 36.7 ± 5 404.7 ± 74 –0.11 Roots Control 22.4 ± 6a 44.4 ± 20a 17.6 ± 4.4a 1.2 ± 0.45 b b b –1 Na2SO4 85.4 ± 30 90.3 ± 3.9 29.9 ± 8.5 4.2 ± 1.5 –1 NaCl 19.3 ± 5a 26.2 ± 4.3c 20.7 ± 2.3a 4.4 ± 3.3 a c a –1 NaCl + Na2SO4 15.1 ± 5 27.7 ± 5.5 20.7 ± 5 0.3 ± 0.1

40 days –0.11 Leaves Control 604.8 ± 140a 80.1 ± 24a 32.7 ± 5.7a 80.6 ± 10a b b ab a –1.88 Na2SO4 103.6 ± 25 39.1 ± 8.8 23.7 ± 6.6 124.3 ± 59 –1.88 NaCl 530.7 ± 214a 38.5 ± 11b 35.5 ± 5.4ac 284.2 ± 41b a ab a c –1.88 NaCl + Na2SO4 385.7 ± 73 57.9 ± 8.7 32.3 ± 10 383.1 ± 15 –0.11 Roots Control 46.7 ± 12a 39.6 ± 8.5 14.9 ± 2.3 1.5 ± 0.5a a a –1.88 Na2SO4 45.8 ± 10 40.6 ± 11 17.1 ± 63.3± 0.7 –1.88 NaCl 24.7 ± 3b 34.5 ± 7 21.9 ± 68.6± 2.8b ab c –1.88 NaCl + Na2SO4 32.9 ± 7 37.9 ± 4.2 19.2 ± 2.3 15.6 ± 3.2

48 days –0.11 Leaves Control 322.2 ± 30 48.9 ± 11a 35.4 ± 4.5a 121.8 ± 57a b ab a –2.6 Na2SO4 335.9 ± 86 85.7 ± 10 40.3 ± 5.4 173.8 ± 66 –2.6 NaCl 527.2 ± 204 32.3 ± 10a 27.9 ± 6ac 258.3 ± 60b c a a –2.6 NaCl + Na2SO4 521.0 ± 61 58.9 ± 16 30.6 ± 8.4 110.2 ± 22 –0.11 Roots Control 44.3 ± 12a 65.6 ± 11 23.3 ± 8.3 5.5 ± 2a b a –2.6 Na2SO4 97.8 ± 22 62.0 ± 23 19.8 ± 5.6 9.8 ± 5.5 –2.6 NaCl 78.2 ± 27ab 49.5 ± 11 21.3 ± 5.7 7.6 ± 3.5a a b –2.6 NaCl + Na2SO4 63.3 ± 30 55.5 ± 17 20.1 ± 3.9 23.1 ± 9.9

Values show the concentration of Putrescine (Put), spermidine (Spm), spermine (Spm) and 1,3 diaminopropane (1,3 Dap). The data are the mean ± SD (n = 6). Means within a column following by different letters indicate statistically significant differences (P < 0.05).

−1.9 MPa), but increased under Na2SO4 and two-salt Discussion treatment at high salinity (Ψo = −2.6 MPa). Spm level in roots was altered only by Na2SO4 Seed germination studies in Prosopis strombulifera treatment at low salinity (Ψo = −1.0 MPa). Spm in showed that effects of salt treatment depend on the leaves showed no difference in salt-treated plants com- osmotic potential and on the type of salt (Sosa et al. pared to control plants, but did show different levels 2005; Llanes et al. 2005). Halophytic plants may not under Na2SO4 vs. NaCl treatment. only tolerate salinity, but in some cases their growth Dap is produced by catabolism of polyamine by is stimulated by NaCl (Flowers et al. 1977). We found polyamine oxidase (PAO). Dap content increased in that shoot growth of P. strombulifera was stimulated roots of NaCl-treated plants at −1.9 MPa, or two-salt by NaCl treatments up to −1.9 MPa (500 mM). Other treated plants at −1.9 MPa and −2.6 MPa. Dap content Prosopis species are less salt tolerant and do not show in leaves was much higher than in roots, with maximal such response; i.e., P. strombulifera is a “true halo- values at low (−1.0 MPa) and moderate (−1.9 MPa) phyte” (euhalophyte). salinity for all treatments. P. strombulifera is less tolerant to Na2SO4 than The uncommon PA cadaverine (Cad) was detected to NaCl, and displays inhibition of shoot growth, and in leaves in only a few cases, and its content was mini- senescence symptoms (chlorosis, necrosis, leaf abscis- mal (data not shown). Cad was found mainly in roots, sion), under Na2SO4 treatment. Similarly, in Cheno- where its content increased with plant age. Only appli- podium rubrum Na2SO4 at −1.6 MPa completely in- cation of high concentration of Na2SO4 induced signif- hibited growth (Warne et al. 1990). In contrast, shoot icantly higher Cad content in roots than for other salt growth of Atriplex prostata seedlings was stimulated by treatments, or control plants (Fig. 4A). Na2SO4 and inhibited by iso-osmotic NaCl (Egan & Root architecture of Na2SO4-treated plants is illus- Ungar 1998). Clearly, effects of different salts on growth trated in Fig. 4B and shows reduction of root length, depend on the plant species. The above studies used and enhanced number of shorter lateral roots. only one-salt solutions, and did not examine the effect Effects of various salt treatments on distribution of two-salt (Na2SO4 and NaCl) mixtures. patterns of PAs are illustrated by pie charts in Fig. 5, P. strombulifera root growth stimulation by NaCl analogously to results for JAs shown in Fig. 3. Con- suggests an adaptive mechanism favoring water and nu- 2− tent of free PAs was clearly higher in leaves than in trient absorption. SO4 anion inhibited root growth in roots, especially in the leaves of NaCl treated plants. In comparison to Cl− anion, and was partially alleviated roots content of PAs was highest under Na2SO4 treat- when both anions were present in the solution. An im- ment. portant finding of the present study is the partial alle- Levels of jasmonates and free polyamines induced by Na2SO4 and NaCl in Prosopis strombulifera 7

Fig. 5. Distribution pattern of free PAs in leaves and roots of control and salt-treated P. strombulifera plants. Control plants (day 48); Na2SO4, NaCl, and two-salt treated plants (Ψo = −2.6 MPa, day 48). Area of circle corresponds to total content of PAs in leaves (top) and roots (bottom). Area of circle in leaves of NaCl-treated plants corresponds to 846.66 nmol/g FW (100%). Areas of circles (and portions thereof) for other plant organs and treatments are proportional.

treatment, in association with chlorophyll a decrease. In NaCl and two salt-treated plants, in contrast, chlorophyll b was decreased, and chlorosis symptoms were not evident, in coincidence with the results reported by Araújo et al. (2006) in the halophyte Atriplex nummularia and Koyro (2006) in Plantago coronopus. Adapta- tions of light harvesting capacity may be used to reduce Fig. 4. (A) Effects of NaCl, Na2SO4, and their two-salt mixture (optimize) photosynthetic efficiency, and consequently (Ψo = −1.0, −1.9, −2.6 MPa) on Cad content in roots of P. reduce oxidative stress. strombulifera. Columns represent average values from four inde- Changes in JAs profiles have been reported in re- pendent experiments, with standard error (S.E.) shown by bars. sponse to abiotic stressors, e.g., NaCl, mannitol/sorbi- (B) Architecture of roots of 40–day-old control and Na2SO4- treated plants (−1.9 MPa). Enlargement: detail of lateral root tol, water stress. In glycophytes (tomato, barley, and formation. Scale bar: 0.5 cm. others), involvement of JAs in response to salt stress is well established (Wasternack & Hause 2002; Pedranzani et al. 2003). 2− viation of SO4 toxicity by two-salt solutions, in terms We show that the same JAs (OPDA, JA, 12-OH- of shoot and root growth, leaf number, leaf area, and JA, 11-OH-JA) found in glycophytes are present in the chlorophyll content. euhalophyte P. strombulifera. Also, large differences in Reduction of leaf area can be considered as total amount of JAs, and in proportion of the com- an avoidance mechanism which minimizes water loss ponents, in roots vs. leaves, and under different salt (Ruiz-Sánchez et al. 2000). Prosopis plants subjected treatments were observed. Miersch et al. (2008) re- to high Na2SO4 concentration decreased total and indi- ported similar differences in JAs content among dif- vidual leaf area at −2.6 MPa, ultimately reaching 30% ferent tissues of various plant species. The major JAs and 65% inhibition respectively. These plants failed to in P. strombulifera roots were OPDA and JA, whereas make efficient osmotic adjustment, leading to negative those in leaves were OPDA and 12-OH-JA. Prevailing osmotic and water potentials, and water imbalance, in of OPDA and JA together with organ-specific patterns leaves (Reginato et al. 2009). of different JA compounds indicate that the concept Leaf chlorophyll content is an important physio- of an “oxylipin signature” (Weber et al. 1997) or “jas- logical index directly related to photosynthesis. High monate signature” (Miersch et al. 2008) can be applied salinity may be associated with suppression of photo- to the halophyte P. strombulifera. synthetic machinery, and reduced chlorophyll content. In leaves, OPDA is synthesized in chloroplasts, and However, we found that chlorophyll content was not sig- there is evidence of a similar pathway in roots (Hause nificantly decreased under low or moderate salt stress. et al. 2000; Abdala et al. 2003). Thus, the high OPDA Only high salinity affected chlorophyll a and b con- content in roots of 29-day-old control P. strombulif- tent. Visible symptoms of early senescence in old leaves era plants suggests active biosynthesis in this organ. (chlorosis, necrosis) were observed only under Na2SO4 P. strombulifera roots decreased total content of JAs 8 M.A. Reginato et al. under high salinity which was an unexpected result. nous Put in Atropabelladonna (Ali 2000), and Put al- OPDA content was higher than that of other JAs, and leviation of growth in salt stressed Brassica juncea by was more affected by salt stress. inducing antioxidative defense system (Verma & Mishra JAs usually inhibit primary root elongation (Was- 2005). Alternatively, the low Spd content in P. strombu- ternack 2007). In P. strombulifera, NaCl-treated plants lifera leaves, under all salt treatments, suggests a rapid showed lowest OPDA content at −1.0 MPa, concomi- degradation of this amine to Put, as part of an intercon- tant with enhanced root growth. The differential re- version pathway well known in animals and also char- sponse of roots vs. leaves to salinity may be attributable acterized in higher plants. Accumulation of Dap and to the root’s function as a “stress-receptor organ”, Put may result from the presence of polyamine oxi- which responds to stress with more dramatic hormonal dases (PAOs) acting on Spd, as reported by Duhazé changes. et al. (2002) in various halophytic species of the genus Content of 12-OH-JA is higher in leaves than in Limonium. The specific effect of Dap is poorly under- roots in both control and treated plants; hence, possibly stood, but may involve biosynthesis of uncommon PAs as a result of an active hydroxylation pathway in leaves, and/or β-alanine, via an oxidative deamination path- or transport of root-generated 12-OH-JA. 11-OH-JA, a way, in some species (Cohen et al. 1998). This is con- permanent component in plants, is usually present in sistent with our finding of several peaks of unknown much lower concentration than that of 12-OH-JA. In P. compounds in our chromatograms (data not shown). strombulifera, salt treatment had no effect on content of Accumulation of common PAs has also been cor- either hydroxylate. Thus, it could be said that JAs are related with formation of adventitious and lateral not directly involved in salt tolerance in this species. roots (Altamura et al. 1991), consistent with our re- Stressful conditions alter the content of endoge- sults (Fig. 4B). Presence of the diamine Cad (1.5- nous PAs, particularly Put, with variation depend- diaminopentane) is sporadic and uncommon. Cad ing on type of stress, plant species, and time of ex- in roots has been associated with cell elongation posure (Ali 2000). Accumulation of PAs in plants is and formation of adventitious roots, similarly to Put very important for their protection against oxidative (Shevyakova et al. 2001; Carrizo et al. 2001). Un- stress induced by abiotic factors. Reliable proof of free der stress conditions, Cad accumulation in plants has PAs functioning as ROS scavengers was obtained in been proposed to compensate a decrease in the con- vitro in a system generating free radicals; NaCl-treated tent of the Put-family PAs; its accumulation in roots of Mesembryanthemum crystallinum plant leaves showed Na2SO4-treated P. strombulifera may be a stress symp- the highest total PA contents and Put was the most tom rather than an adaptative response. Biosynthesis of abundant PA (Shevyakova et al. 2006). Put in P. strom- uncommon PAs such as 1.3 Dap, Cad, and others, has bulifera was highest in roots of Na2SO4-treated plants been associated with the capacity of some organisms at −1 MPa which also showed significant increases in to grow or function under extreme conditions (Flores Spd and Spm, indicating that Na2SO4 causes a pro- 1991). nounced oxidative stress, confirmed by higher malondi- From these results we conclude that: (1) the ma- aldehyde (MDA) levels in these plants than those un- jor salts present in soils of central Argentina (and sev- der NaCl treatment (unpublished data). This salt is not eral other countries) cause differential biochemical and stressful for this species up to 500 mM. physiological plant responses, (2) P. strombulifera is a Low Put content in leaves of Na2SO4-treated P. “true halophyte” (euhalophyte) only for NaCl being strombulifera is correlated with inhibition of shoot much less tolerant to Na2SO4, (3) an important finding 2− growth at −1.9 and −2.6 MPa, and possibly associated is the partial alleviation of SO4 toxicity by treatment 2− with a general metabolic alteration caused by SO4 with two-salt mixture (4) JAs are not directly involved anion (Reginato 2009). Similarly, growth inhibition of in salt tolerance in this species, (4) beneficial effects Fraxinus angustifolia callus under NaCl stress was asso- of Put accumulation in NaCl treated plants maybe in- ciated with reduction of endogenous PAs, particularly ferred, probably associated with the antioxidative de- Put (Tonon et al. 2004). fense system (5) another novel finding is the accumu- A stress-induced activation of the complete path- lation of the uncommon PA cadaverine in roots under 2− way of PA biosynthesis with the accumulation of Spd high Na2SO4, which may be related to SO4 toxicity. 2− and Spm is observed relatively infrequently (Tiburcio et The mechanism of SO4 toxicity at cellular level al. 1994). It has been suggested that NaCl suppresses is not yet known, and it is currently under study in our the key enzyme of Spd and Spm biosynthesis, SAM de- laboratory. carboxylase. Therefore, the biosynthesis of these two PAs is retarded and its precursor, Put, accumulates ac- Acknowledgements tively (Kuznetzov & Shevyakova 2007). Thus, the maintenance of Put content in leaves of NaCl-treated plants This study was supported with funds from CONICET PIP No 5628, PICTO-ANPCYT-UNRC No 30093, SECYT- in the present study may involve a similar mechanism Universidad Nacional de Río Cuarto and Ministerio de and a role for Put in NaCl tolerance inP. strombulifera. Ciencia y Tecnología de la Provincia de Córdoba (R No In this sense, beneficial effects of Put on NaCl treated 1210/2007-2011), Argentina, to V. Luna, and a fellowship plants have been reported, such as stimulation of ger- from CONICET to M. Reginato. We also thank Dr. Steve mination, seedling growth, alkaloid content and endoge- Anderson for language edition. Levels of jasmonates and free polyamines induced by Na2SO4 and NaCl in Prosopis strombulifera 9

References Marcé M., Brown D., Capell T., Figueras X., Tiburcio A. 1995. Rapid high liquid chromatographic method for the quantita- Abdala G.I., Miersch O., Kramell R., Vigliocco A., Agostini E., tion of polyamines as their dansil derivatives. Applications to 666: Forchetti G. & Alemano S. 2003. Jasmonate and octade- plant and animal tissues. J. Chromat. B. 329–335. canoid occurrence in tomato hairy roots. Endogenous level Miersch O., Neumerkel J., Dippe M., Stenzel I. & Wasternack changes in response to NaCl. Plant Growth Regul. 40(1): C. 2008. Hydroxylated jasmonates are commonly occurring 21–27. metabolites of jasmonic acid and contribute to a partial 177: Ali R. 2000. Role of putrescine in salt tolerance of Atropa bel- switch-off in jasmonate signaling. New Phytol. 114–127. Pedranzani H., Racagni G., Alemano S., Miersch O., Ramírez ladonna plant. Plant Sci. 152: 173–179. I., Peńa-Cortés H., Taleisnik E., Machado-Domenech E. & Altamura M., Torrigiani P., Capitani F., Scaramagli S. & Bagni Abdala G. 2003. Salt tolerant tomato plants show increased N. 1991. De novo root formation in tobacco thin layers af- levels of jasmonic acid. Plant Growth Regul. 41: 149–158. fected by inhibition of polyamine biosynthesis. J. Exp. Bot. 42: Reginato M., Llanes A., Reinoso H., Moschetti E. & Luna V. 1575–1582. 2009. Foliar area, stomatal caracteristics and water relations Araújo S.A.M., Silveira J.A.G., Almeida T.D., Rocha I.M.A., in the halophyte Prosopis strombulifera under salinity. Biocell Morais D.L. & Viégas R.A. 2006. Salinity tolerance of halo- 33: 254. phyte Atriplex nummularia L. grown under increasing NaCl Reginato M.A. 2009. Respuesta de la halófita Prosopis strom- 10: levels. Rev. Bras. Eng. Agríc. Amb. 848–854. bulifera a diferentes medios salinos. Modificaciones de los Bressan R., Zhang C., Zhang H., Hasegawa P., Bohnert H. & Zhu parámetros morfofisiológicos y su regulación hormonal. PhD J-K. 2001. Learning from the Arabidopsis experience. The Thesis, Departamento de Ciencias Naturales, Universidad next gene search paradigm. Plant Physiol. 127: 1354–1360. Nacional de Río Cuarto, Córdoba, Argentina. Carrizo C., Pitta-Alvarez S., Kogan M., Giulietti A. & Tomaro M. Reinoso H., Sosa L., Ramirez L. & Luna V. 2004. Salt-induced 2001. Occurrence of cadaverine in hairy roots of Brugmansia changes in the vegetative anatomy of Prosopis strombulifera candida. Phytochem. 57: 759–763. (Leguminosae). Can. J. Bot. 82: 618–628. Cohen S.S. 1998. A Guide to the Polyamines. Oxford University Reinoso H., Sosa L., Reginato M. & Luna V. 2005. Histologi- Press, Oxford. cal alterations induced by sodium sulfate in the vegetative Duhazé C., Gouzerh G., Gagneul D., Larher F. & Bouchereau anatomy of Prosopis strombulifera (Lam.) Benth. World J. 1: A. 2002. The conversion of spermidine to putrescine and 1,3 Agric. Sci. 109–119. diaminopropane in the roots of Limonium tataricum.Plant Ruiz-Sánchez M., Domingo R., Torrecillas A. & Pérez-Pastor A. Sci. 163: 639–646. 2000. Water stress preconditioning to improve drought resis- tance in young apricot plants. Plant Sci. 156: 245–251. Egan T. & Ungar I. 1998. Effect of different salts of sodium and Santa Cruz A., Acosta M., Rus A. & Bolarin M. 1999. Short- potassium on the growth of Atriplex prostata (Chenopodi- term salt tolerance mechanisms in differentially salt tolerant aceae). J. Plant Nutr. 21(10): 2193–2205. tomato species. Plant Physiol. Biochem. 37: 65–71. Felker P. 2007. Unusual physiological properties of the arid Shevyakova N., Rakitin V., Duong D., Sadomov N. & Kutnetzov adapted tree legume Prosopis and their applications in de- V. 2001. Heat shock-induced cadaverine accumulation and veloping countries, pp. 1–41. In: De la Barrera E. & Smith translocation throughout the plant. Plant Sci. 161: 1125– W. (eds), Perspectives in Biophysical Plant Ecophysiology: A 1133. Tribute to Park Nobel, Mildred E. Mathias Botanical Garden Shevyakova N., Rakitin V., Stetsenko L., Aronova E. & Kuzn- University of California, Los Angeles. ersov V. 2006. Oxidative stress and fluctuations of free and Flores H.E. 1991. Changes in polyamine metabolism in response conjugated polyamines in the halophyte Mesembryanthemum to abiotic stress, pp. 214–255. In: Slocum R. & Flores H (eds), crystallinum L. under NaCl salinity. Plant growth Regul. 50: The biochemistry and physiology of polyamines in plants, 69–78. Boca Raton CRC Press, Florida. Sosa L., Llanes A., Reginato M., Reinoso H. & Luna V. 2005. Os- Flowers T., Troke P. & Yeo A. 1977. The mechanism of salt tol- motic and Specific Ion Effects on the Germination of Prosopis erance in halophytes. Ann. Rev. Plant Physiol. 28: 89–121. strombulifera (Lam.) Benth. Ann. Bot. 96: 261–297. Gong Q., Li P., Ma S., Rupassara S. & Bohnert H. 2005. Salin- Tiburcio A., Besford R., Capell T., Borell A., Testillano T. & ity stress adaptation competence in the extremophile Thel- Risueño M. 1994. Mechanism of polyamine action during lungiella halophila in comparison with its relative Arabidopsis senescence responses induced by osmotic stress. J. Exp. Bot. thaliana.PlantJ.44: 826–839. 45: 1789–1800. Hause B., Stenzel I., Miersch O., Maucher H., Kramell R., Ziegler Tonon G., Kevers C., Faivre-Rampant O., Graziani M. & Gaspar J. & Wasternack C. 2000. Tissue-specific oxylipin signature T. 2004. Effect of NaCl and mannitol iso-osmotic stresses on of tomato flowers: Allene oxide cyclase is highly expressed proline and free polyamine levels in embryogenic Fraxinus in distinct flower organs and vascular bundles. Plant J. 24: angustifolia callus. J. Plant Physiol. 161: 701–708. 113–126. Verma S. & Mishra S. 2005. Putrescine alleviation of growth in Koyro H-W. 2006. Effect of salinity on growth, photosynthesis, salt stressed Brassica juncea by inducing antioxidative de- 162: water relations and solute composition of the potential cash fence system. J. Plant Physiol. 669–677. crop halophyte Plantago coronopus (L.). Environ. Exp. Bot. Warne P., Guy R., Rollins L. & Reid D. 1990. The effects of 56: 136–146. sodium sulphate and sodium chloride on growth, morphol- ogy, photosynthesis, and water use efficiency of Chenopodium Krishnamurthy R. & Bhagwat K. 1989. Polyamines as modu- rubrum.Can.J.Bot.68: 999–1006. lators of salt tolerance in rice cultivars. Plant Physiol. 91: Wasternack C. & Hause B. 2002. Jamonates and octadecanoids: 500–504. signals in plant stress responses and development. Prog. Nuc. Kutznetsov V. & Shevyakova N. 2007. Polyamines and stress tol- Acid Res. Mol. Biol. 72: 165–221. 1: erance of plants. Plant Stress 50–71. Wasternack C. 2007. Jasmonates: An Update on Biosynthesis, Legocka J.& Kluk A. 2005. Effect of salt and osmotic stress on Signal Transduction and Action in Plant Stress Response, changes in polyamine content and arginine decarboxylase ac- Growth and Development. Ann. Bot. 100: 681–697. tivity in Lupinus luteus seedlings. J. Plant Physiol. 162: 662– Weber H., Vick B. & Farmer E. 1997. Dinor-oxo-phytodienoic 668. acid: A new hexadecanoid signal in the jasmonate family. Liu J., Kitashiba H., Wang J., Ban Y. & Moriguchi T. 2007. Proc. Nat. Acad. Sci. USA 94: 10473–10478. Polyamines and their ability to provide environmental toler- Received June 28, 2011 24: ance to plants. Plant Biotech. 117–126. Accepted January 13, 2012 Llanes A., Reinoso H. & Luna V. 2005. Germination and early growth of Prosopis strombulifera seedlings in different saline solutions. World J. Agric. Sci. 1: 120–128.