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 concen- trations (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 in- volved 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.