Alleviation of salinity stress in broccoli using foliar urea or methyl-jasmonate: analysis of growth, gas exchange, and isotope composition
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We studied the effects of foliar application of urea or methyl-jasmonate (MeJA) on the salinity tolerance of broccoli plants (Brassisca oleracea L. var. italica). Plant dry weight, leaf CO2 assimilation, and root respiration were reduced significantly under moderate saline stress (40 mM NaCl) but application of either urea or MeJA maintained growth, gas exchange parameters, and leaf N–NO3 − concentrations at values similar to those of non-salinized plants. Additionally, when these two foliar treatments were applied leaf Na+ concentration was reduced compared with control plants grown at 40 mM NaCl. However, at a higher salt concentration (120 mM NaCl), no effect of the foliar applications was found on these parameters. Salinity also decreased leaf δ15N but increased δ13C. Our study shows the feasibility of using foliar urea or MeJA to improve tolerance under moderate saline stress.
KeywordsBroccoli Foliar application Urea Isotope composition Methyl-jasmonate Salinity
Salinity is a major environmental factor limiting plant growth and crop productivity, and salinization of irrigated and surrounding areas in the arid tropics and sub-tropics has not diminished (Tuna et al. 2008b). The loss of farmable land due to salinisation is in direct conflict with the growing population, posing a major challenge for maintaining world food supplies (Purty et al. 2008). The reduction in plant growth and yield is mainly due to an osmotic effect of the accumulation of salts near the root zone whereas the build up of toxic saline ions in plant tissues is responsible for the progressive impairment of several physiological processes (Munns 2002). In a saline environment, ion homeostasis can be disturbed by excessive uptake of Na+ and Cl−. Competition between these and other anions and cations are well documented and may result in reduced growth (De Pascale et al. 2005). Thus, in saline environments, plants have developed different adaptative mechanisms (Borsani et al. 2003). One adaptive plant response to salt stress is synthesis and accumulation of low-molecular weight organic compounds in the cytosol and organelles (Bartels and Sunkar 2005). Compatible osmolytes reported to be affected by salinity stress include simple sugars, alcohols or polyols, amino acids, quaternary amino acid derivatives, and sulfonium compounds (Zhu 2001).
Salinity can reduce N accumulation in plants (Pessarakli 1991) and an increase in Cl− uptake and accumulation is often accompanied a decrease in NO3 − concentration. Many authors attributed this reduction to Cl− antagonism of NO3 − uptake (Kafkafi et al. 1992; Bar et al. 1997). Inhibition of nitrogen uptake may occur by NO3 −/Cl− interaction at the sites of ion transport (Cram 1983), because sodium results in severe membrane depolarization in plants (Suhayda et al. 1990)—which has been linked to non-competitive inhibition of nitrate uptake (Hawkins and Lewis 1993). However, the method of N application (via soil or leaves) influences growth and storage of N (Habib et al. 1993); thus, when N supply to the roots is impaired, applications of foliar urea can be used effectively (Nicoulaud and Bloom 1996; del Amor et al. 2009). Urea is one of the most widely-used foliar-N fertilizers, characterized by high leaf penetration rate and low cost and most plants can absorb rapidly and hydrolyze urea in the cytosol (Witte et al. 2002). Urea can also increase the level of storage N compounds such as amino acids and proteins (Dong et al. 2004); thus, foliar urea could directly affect N metabolism under saline conditions and therefore amino acids synthesis. Moreover, the application of fertilizer N directly to leaves, especially urea, can be a potential alternative to conventional soil fertilization and does not contribute to soil salinity or potential groundwater contamination (Embleton et al. 1986).
In addition to the foliar supply of N-fertilizer, exogenous application of some phytohormones could also affect directly the plant response to salinity (Tuna et al. 2008a). Jasmonates are ubiquitously-occurring lipid-derived compounds with signal functions in plant responses to abiotic and biotic stresses, as well as in plant growth and development (Wasternack 2007), and recent studies pointed out a number of salinity and jasmonates-regulated genes induced by wounding (Cheong et al. 2002). Applied exogenously, they can induce physiological changes identical to characteristic parts of the stress responses (Tsonev et al. 1998). Thus, jasmonates have been identified as stress modulators suppressing or enhancing the stress responses of plants. Jasmonate levels were increased with high salinity in Iris hexagona (Wang et al. 2001) and rice, resulting in the induction of genes involved in stress-related jasmonates biosynthesis (Tani et al. 2008). Fedina and Benderliev (2000) found that exogenously-applied MeJA supplied simultaneously with NaCl helped algae to counteract salt stress; therefore, the toxic ion effects due to salinity could cause membrane damage and hence trigger the release of the lipid pre-cursors for jasmonate synthesis. Moreover, Parra-Lobato et al. (2009) concluded that exogenous MeJA may be involved in the oxidative stress processes by regulating antioxidant enzyme activities. These results suggest a role for MeJA in the plant response to saline stress. However, relatively little is known about its involvement in the response to abiotic stress in broccoli, especially salinity stress.
The analysis of the natural abundances of stable carbon isotopes in plant dry matter (δ13C) provides information on the long-term water use efficiency (WUE) of plants, and values of δ13C are determined mainly by three processes: diffusion of CO2 through stomata, CO2 assimilation by carboxylase, and metabolism of compounds (Farquhar et al. 1989). In contrast to gas exchange techniques that provide measurements at a single point in time, the leaf carbon isotopic composition integrates the ratio of the intercellular to the ambient CO2 concentrations for longer periods (all the saline-stress period). The basis of the biochemical discrimination against 13C in C3 plants lies with the primary carboxylating enzyme (Rubisco), which discriminates against 13C because of the intrinsically lower reactivity of 13C compared with 12C (Brugnoli and Farquhar 2000). δ13C also gives information about how the nitrogen is used by the plant under water and nutritional (N) stress conditions (Cabrera-Bosquet et al. 2007). Additionally, other studies considered the natural abundance of δ15N as an index of genotypic performance under given stress conditions, specifically water stress (Robinson et al. 2000) and salinity (Handley and Scrimgeour 1997). Carbon stable isotope composition (δ13C) is seen as an index of the integrated response of a physiological characteristic to environmental factors (Choi et al. 2005). However, little is known about correlations between stable isotope composition and salinity in broccoli, especially when foliar treatments are applied.
Broccoli is a crop moderately-sensitive to salinity but has greater salt tolerance than most other common vegetables including melons, corn, lettuce, peppers, onions, and carrots (LeStrange et al. 1996). Salt stress induced detrimental effects on growth and yield depend on several factors such as the level of stress experienced, the time of exposure to the stress, and the soil physical–chemical properties (Maas and Grattan 1999). Additionally, broccoli is a nitrogen-demanding crop and N supply is a determining factor for its productivity and quality (Everaarts and De Willigen 1999); a lower supply of nitrate to growing leaves may be responsible for the inhibition of growth under saline conditions (Hu and Schmidhalter 1998). Therefore, foliar application of fertilizers could alleviate or neutralize growth inhibition due to salinization (Sultana et al. 2001).
The aim of this study was to test the hypothesis that exogenous, foliar application of N-fertilizer (urea) or a phytohormone (methyl-jasmonate) could diminish or counteract the metabolic imbalance produced by a high NaCl concentration in the nutrient solution. We aimed to provide an insight into the physiological significance and usefulness of δ13C and δ15N signatures under saline conditions and to elucidate their responses when foliar N-fertilizer or a phytohorme are applied to broccoli plants.
Materials and methods
Plant material, growth conditions and treatments
Broccoli plants (Brassisca oleracea L. var. italica cv. Lord) were grown in 12-L black containers filled with coconut coir fiber. Plants were irrigated with a solution with the following composition in meq L−1; NO3 −: 12.0; H2PO4 −: 1.0; SO4 2−: 7.0; K+: 7.0; Ca2+: 9.0; Mg2+: 4.0. Irrigation was supplied by self-compensating drippers (2 lh−1) and fresh nutrient solution was applied to avoid salt accumulation, with a minimum of 35% drainage. Plants were grown in a climate chamber designed by our department specifically for plant research proposes (del Amor et al. 2010), with fully-controlled environmental conditions: 70% RH, 16/8 h day/night photoperiod, 22/18°C, and a photosynthetically-active radiation (PAR) of 250 μmol m−2 s−1 provided by a combination of fluorescent lamps (Philips TL-D Master reflex 830 and 840) and high-pressure sodium lamps (Philips Son-T Agro). Fifteen days after transplanting (DAT) treatments were begun by adding NaCl to the nutrient solution (40 or 120 mM) and spraying foliar urea (10 gl−1) or methyl-jasmonate (5 mM) onto the plants. Triton X-10 (0.125%) and ethanol (0.04%) were added to the foliar applications as surfactant and solvent respectively. Hormonal treatment was applied once, 11 DAT, whilst foliar urea was applied every 2 weeks. To avoid treatment contamination, treatments were sprayed independently outside the climate chamber and plants were left there until complete evaporation of the sprayed solution (about 1 h). Nine treatments were analyzed (0, 40, and 120 mM NaCl, non-sprayed or sprayed with foliar urea or Me-JA). At harvest (60 DAT), 45 plants (5 plants per treatment) were analyzed, and the aerial parts weighed and separated into leaves and stems (including petioles). Dry weight samples were determined after a minimum of 96 h at 65°C.
Leaf Cl−, NO3 −, and Na+ concentrations were determined in dry matter, Cl− and N-NO3 − by Capillary Electrophoresis (Waters, Capillary Ion Analyzer) and Na+ by inductively coupled plasma–optical emission spectrometer (ICP-OES, Varian Vista MPX).
At the end of the experimental period, net CO2 assimilation was measured in the youngest fully-expanded leaf of each plant, using a CIRAS-2 (PP system) with a PLC6 (U) Automatic Universal Leaf Cuvette. The cuvette provided light (LED) with a photon flux of 800 μmol m−2 s−1, 380 ppm CO2, and a leaf temperature of 22°C. Soil (root) respiration was measured for each plant with the CIRAS-2 and the SRC-1 Soil Respiration Chamber.
Dry, powdered leaf samples were packed in tin capsules and analyzed by isotope ratio mass spectrometry (Continuous Flow Isotope Ratio Mass Spectrometer-CF-IRMS, Micromass Isoprime, Eurovector). The N content was expressed as a percentage of dry matter. The 13C/12C ratios were expressed in δ notation determined by: δ13C (‰) = [(13C/12Csample) − (13C/12Cstandard)]/(13C/12Cstandard) × 1,000 (Farquhar et al. 1989), where ‘sample’ refers to plant material and ‘standard’ refers to Pee Dee Belemnite (PDB) calcium carbonate. The same δ notation was used for the 15N/14N ratio expression (δ15N) but with ‘standard’ referring to air. Isotope secondary standards of known 13C/12C and 15N/14N ratios were used for calibration (USGS-24 and urea, respectively).
Data were tested first for homogeneity of variance and normality of distribution, and were analyzed through an analysis of variance using the Generalized Linear Model (GLM) procedure of SPSS (version 13.0). Additionally, Duncan′s multiple range test was used to determine the significance of differences (P ≤ 0.05). The number of replicates for each treatment was five.
Results and discussion
Plant growth, mineral content, and gas exchange
Our data show that supplemental foliar spray of urea to the salinized plants appeared to partially counteract the deleterious effects of salinity. Hasaneen et al. (2008) found a similar effect in lettuce but the magnitude of response was most pronounced with higher urea concentrations. Additionally, Sultana et al. (2001) found that foliar spray of fertilizers partially minimized the salt-induced nutrient deficiency and increased photosynthesis, photosynthesis-related parameters, yield and yield components. Tsonev et al. (1998) reported that pre-treatment of barley plants with JA before salinisation ameliorated the inhibitory effects of salinity on growth when compared to a direct salt stress. However our data show that this nullification of the harmful effects of salinity in broccoli plants by urea or MeJA was only effective at low salt concentration.
Therefore, the inhibition of nitrogen uptake observed in treatments not involving foliar application, due to salinity-imposed competition with Cl− (Papadopoulos and Rending 1983), was successfully overcome at moderate salinity, but not at high salinity. López-Berenguer et al. (2007) found that a NaCl concentration higher than 60 mM exceeded the threshold of resistance in broccoli plants. But the reduced N–NO3 − concentration in the leaves at higher salinity could be strongly affected by the higher osmotic effect of the nutrient solution, reducing N uptake.
Foliar urea or MeJA did not improve root respiration under non-saline conditions, but irrigation with 40 mM NaCl increased soil respiration by 36.4 and 29.4% when urea or MeJA was applied, compared with the non-sprayed plants under the same growth conditions. However, a non-beneficial effect was found at 120 mM NaCl. At high salinity, salts can build up in leaves to excessive levels, but exactly how the salts exert their toxicity remains unknown. Salts may build up in the apoplast and dehydrate the cell, they may build up in the cytoplasm and inhibit enzymes involved in carbohydrate metabolism, or they may build up in the chloroplast and exert a direct toxic effect on photosynthetic processes (Munns and Tester 2008).
Chloride build up in leaves of plants can produce a progressive decrease in plant growth and a reduction in photosynthesis and transpiration (López-Climent et al. 2008; Zheng et al. 2008). However, as reported by Walia et al. (2007), the treatment with JA partially alleviated photosynthetic inhibition caused by moderate salinity stress, and genes involved in JA biosynthesis show increased abundance (Walia et al. 2006). Additionally, foliar application of urea also appeared to counteract the stress-induced damage, maintaining growth and photosynthesis at moderate stress. Recently, Hasaneen et al. (2008) found a similar effect for NaCl-stressed lettuce, thus indicating that toxic ions, such as Na+ in the leaves, may interfere with phloem loading, restricting the movement of nutrients from roots to shoot (Sultana et al. 2001). Therefore, N applied directly to the leaves decreases the inhibition due to the toxic effects of Na+ and Cl− or minimizes the salinity-induced nutrient deficiency, maintaining the NO3 − concentration.
Carbon and nitrogen isotope composition
Foliar δ 15N values also show a strong relationship with water availability (Handley et al. 1999): in our experiment, this may be the result of the osmotic effect of the saline solution, and these values may reflect changes in N allocation within leaves (Stock and Evans 2006).
Values of δ 13C are determined mainly by three processes: diffusion of CO2 through stomata, CO2 assimilation by carboxylase, and metabolism of compounds (Wei et al. 2008). The data showing depression of photosynthesis may be attributed to stomatal closure, increase in water use efficiency, and non-stomatal factors such as damaged cell membranes, reduced mesophyll conductance, and decreased Rubisco activity (Sobrado 1999), but δ 13C could be used as one of the key limiting factors of photosynthesis (Brugnoli and Lauteri 1991). Thus, if stomatal limitation was the main cause of photosynthetic inhibition, leaf δ 13C would increase with increasing salinity, whereas if non-stomatal factors became the key limitation, the values would tend to decrease (Wei et al. 2008). Therefore, our results indicate a stomatal limitation of photosynthesis in broccoli plants due to salinity.
In conclusion this study shows (1) the feasibility of using both foliar urea or methyl-jasmonate to increase salinity tolerance as growth, photosynthesis, and root respiration were not impaired under moderate saline stress (2) that foliar treatments maintained NO3 − and reduced Na+ concentrations in the leaves at moderate salinity, and (3), a significant relationship for δ 13C and δ 15N, with salinity: δ 13C values increased whilst δ 15N decreased–which may be attributable to stomatal adjustment and changes in N allocation within leaves, respectively.
Paula Cuadra-Crespo is the recipient of a pre-doctoral fellowship from the IMIDA. The authors thank G. Ortuño for his technical assistance. This work has been supported by the Instituto Nacional de Investigaciones Agrarias (INIA), through project RTA2008-00089. Part of this work was also funded by the European Social Fund.
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