Journal of Plant Nutrition Nitrogen, Phosphorus, and

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This article was downloaded by: [Centro Edaf Bio Aplicada Segura] On: 12 March 2015, At: 03:49 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Plant Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpla20

Nitrogen, Phosphorus, and Sulfur Nutrition in Broccoli Plants Grown Under Salinity a

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Carmen López-Berenguer , Micaela Carvajal , b

Cristina Garcéa-Viguera & Carlos F. Alcaraz

a

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Departamento de Nutrición Vegetal , CEBAS-CSIC , Murcia, Spain b

Departamento de Ciencia y Tecnología de los Alimentos , CEBAS-CSIC , Murcia, Spain Published online: 03 Dec 2007.

To cite this article: Carmen López-Berenguer , Micaela Carvajal , Cristina GarcéaViguera & Carlos F. Alcaraz (2007) Nitrogen, Phosphorus, and Sulfur Nutrition in Broccoli Plants Grown Under Salinity, Journal of Plant Nutrition, 30:11, 1855-1870, DOI: 10.1080/01904160701629062 To link to this article: http://dx.doi.org/10.1080/01904160701629062

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Journal of Plant Nutrition, 30: 1855–1870, 2007 Copyright © Taylor & Francis Group, LLC ISSN: 0190-4167 print / 1532-4087 online DOI: 10.1080/01904160701629062

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Nitrogen, Phosphorus, and Sulfur Nutrition in Broccoli Plants Grown Under Salinity Carmen L´opez-Berenguer,1 Micaela Carvajal,1 Cristina Garc´ıa-Viguera,2 and Carlos F. Alcaraz1 1

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Departamento de Nutrici´on Vegetal, CEBAS-CSIC, Murcia, Spain Departamento de Ciencia y Tecnolog´ıa de los Alimentos, CEBAS-CSIC, Murcia, Spain

ABSTRACT Broccoli (Brassica oleracea L. var. ‘Italica’) is a recognized health-promoting vegetable and shows a moderate sensitivity to salinity. As very little is known about the effect of salt stress on broccoli plants, the objective was to evaluate nitrogen (N), phosphorous (P), and sulfur (S) nutrition in plants grown under saline conditions. For this objective, the contents of nitrate, phosphate, and sulfate, and total nitrogen, phosphorus, and sulfur, as well as related metabolic enzymes, were determined for plants grown with 0, 20, 40, 60, 80, or 100 mM sodium chloride (NaCl) for two weeks. Nitrate, phosphate, and sulfate concentration in leaves and roots showed a maximum at 40–60 mM NaCl. Up to these salt levels, broccoli plants showed a normal development, but over these salt levels, broccoli plants showed a decrease of nitrate reductase and an increase of the acid phosphatase. From 60 to 100 mM NaCl, the nutritional disorders indicated that the threshold of resistance was exceeded. Keywords: Broccoli, salt stress, nitrogen, phosphorous, sulfur, nitrate reductase, acid phosphatase, O-acetylserine (thiol) lyase

INTRODUCTION Salinity is the most serious threat to agriculture and the environment in many parts of the world. Under salinity, multiple adverse phenotypic expressions of injury occur, which affect plant growth and development at the Received 17 April 2006; accepted 10 March 2007. Address correspondence to Micaela Carvajal, Departamento de Nutrici´on Vegetal, CEBAS-CSIC. P.O. Box 164, 30100 Espinardo, Murcia, Spain. E-mail: mcarvaja@ cebas.csic.es 1855

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physiological and biochemical levels (Munns, 2002). The injurious effects of salinity on plant growth involve (1) low osmotic potential of the soil solution (water stress), (2) nutritional imbalance, (3) specific ion effect (salt stress), or (4) a combination of these factors (Marschner, 1995; Shannon, 1998). Nitrogen (N) is the fourth most abundant element in living organisms and nitrate is the major source of nitrogen for plants, serving both as a nutrient and a signal and having important effects on plant metabolism and growth (Crawford et al., 2000). Nitrate uptake and transport seem to be particularly sensitive to salt stress. Between chloride (Cl− )and nitrate there exists an interactive effect (Hu and Schmidhalter, 2005). Chloride competes with nitrate for uptake and translocation within plants by transporter proteins (Campbell, 1999). The sensitivity of different species to salt is related to the chloride sensitivity of their uptake systems (Leidi and Lips, 1990). The loading of nitrate into root xylem is a highly salt-sensitive step as well (Peuke et al., 1996; Speer et al., 1994; Tischner, 2000). Salinity may affect strongly the overall nitrate assimilation process, because nitrate is required to induce nitrate reductase (NR), the key enzyme of the overall assimilation process (Campbell, 1999). Nitrate reductase activity in leaves is largely dependent on the nitrate flux from roots (Ferrario-Mery et al., 1998; Foyer et al., 1998) and is affected severely by sodium chloride (NaCl) salt stress (Abd-El Baki et al., 2000; Silveira et al., 2001). Nitrate reductase is regulated at the transcriptional, translational, and post-translational levels (Tischner, 2000). Phosphorus (P) plays a role in an array of processes, including energy generation, nucleic acid synthesis, photosynthesis, glycolysis, respiration, membrane synthesis and stability, enzyme activation/inactivation, redox reactions, signalling, carbohydrate metabolism, and nitrogen fixation. Therefore, its assimilation, storage and metabolism are very important for plant growth and development (Vance et al., 2003). But, although bound phosphorus is quite abundant in many soils, it is largely unavailable for uptake; for this reason, phosphorus is frequently the most limiting element for plant growth and development (Vance et al., 2003). Phosphorus is preferentially taken up by plants in its orthophosphate forms (Duff et al., 1994). Phosphatases have been classified traditionally as alkaline or acid phosphatases according to whether their optimal pH for catalysis is above or below pH 7.0 (Vincent et al., 1992). The intracellular acid phosphatases (AP) are responsible for the P-hydrolysis from organic compounds, favoring P mobilization and translocation from senescent tissues (Duff et al., 1994; Lefebvre et al., 1990). Therefore, this enzyme activity is a physiological characteristic related to plant efficiency in P acquisition and utilisation (Tadano et al., 1993). It has been suggested that plants with lower root or leaf AP activities would have adequate or sufficient concentrations of phosphates in tissues, even under low external-phosphorus conditions, when compared to plants

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with higher AP activity in the same conditions (Machado and Furlani, 2004; Raposo et al., 2004; Tadano et al., 1993). The interaction between salinity and P nutrition is particularly complex. Plant responses depend largely on the species or cultivar being examined, the stage of plant growth, the level of NaCl, the form of P, and the environmental conditions of the experiment (Loupassaki et al., 2002; Rogers et al., 2003; Villora et al., 2000). The diversity of results obtained seems to depend, to some extent, on whether the studies were conducted in the field or in solution culture (Hu and Schmidhalter, 2005). The exact mechanism by which NaCl influences P uptake is unknown. Physical and chemical changes at the membrane level and the suppression of P uptake and accumulation in shoots have been suggested to be the result of competition with Cl− (Papadopoulos and Rendig, 1983). On the other hand, it has been reported that AP activity increases in salt-stress conditions (Parida and Das, 2004; Szabonagy et al., 1992). Sulfur (S) is an essential macronutrient required for plant growth and development and is available to plants primarily in the form of anionic sulfate present in soil (Leustek and Saito, 1999). Sulfate is transported actively into roots, distributed, mostly non-metabolised, throughout the plant (Leustek and Saito, 1999) and then assimilated into cysteine in different organs of the plant, but preferably in leaves (Romero et al., 2001). Cysteine is used for direct protein and glutathione synthesis, or functions as an S-donor for methionine and secondary metabolite biosynthesis (Leustek and Saito, 1999). During saline stress, there is a high demand for cysteine, which is in agreement with the role of sulfuric compounds as osmolytes or antioxidants (Barroso et al., 1999). The integration of reduced sulfur into the amino acid cysteine, a central step in the assimilation of inorganic sulfur, is catalysed by O-acetylserine (thiol) lyase (OASTL) and requires two substrates: free sulfide, provided by the sulfate-reduction pathway, and Oacetylserine, an energetically-activated derivative of serine that is metabolically unique to cysteine synthesis (Wirtz et al., 2004). It has been reported that exposure to salt-stress conditions induced higher rates of cysteine synthesis as a result of an increased expression of the cytosolic form of OASTL (Barroso et al., 1999; Fediuc et al., 2005; Romero et al., 2001), and also it could be that this enzyme is related with salt tolerance (Fediuc et al., 2005; Romero et al., 2001) . Broccoli (Brassica oleracea L. var. Italica) is a recognized healthpromoting vegetable and is moderately sensitive to salinity, although it has higher tolerance than other common vegetables such as lettuce, onion, maize and carrot (Shannon and Grieve, 1999). As very little is known about the effect of salinity on broccoli plants, this work was carried out to study the effect of salinity on nitrate, sulfate, and phosphate nutrition and on their relationship with assimilation- or metabolism-related enzymes.

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MATERIALS AND METHODS

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Plant Material and Growth Conditions Broccoli seeds (Brassica oleracea var. italica, cv. ‘Marathon’) were prehydrated with aerated, de-ionised water for 12 h and germinated in vermiculite, at 28◦ C in an incubator, for 2 d. They were then transferred to a controlledenvironment chamber with a 16 h light and 8 h dark cycle and 25◦ C day and 20◦ C night air temperatures. The relative humidity (RH) was 60% (day) and 80% (night) and photosynthetically-active radiation (PAR) was 400 µmol m−2 s−1 , provided by a combination of fluorescent tubes (Philips TLD 36 W/83, Germany and Sylvania F36 W/GRO, USA) and metal-halide lamps (Osram HQI.T 400 W, Germany). Five days after transfer to controlled-environment chamber, the seedlings were placed in 15-L containers with continuouslyaerated Hoagland nutrient solution. The solution was replaced completely once a week. After 14 d when plants were 19-d-old), plants were treated with different NaCl concentrations: 0 (control), 20, 40, 60, 80, or 100 mM NaCl. Mineral contents were determined and enzyme assays performed after 14 d of treatment application, when plants were 33-d-old. The harvests were all performed in the middle of the light period.

Mineral Content Analysis Leaves and roots collected 14 d after the salt treatments were dried at 65◦ C for 5 d. A Dionex D-100 ion chromatograph, with an ionpac AS 124-4 mm (10–32) column and an AG 14 (4 × 50 mm) guard column, was used to measure ion concentrations in leaves and roots. The flow rate was adjusted to 1 mL min−1 , with an eluent composition of 0.5 mM sodium carbonate (NaCO3 ) and 0.5 mM sodium bicarbonate (NaHCO3 ). Ion concentrations were calculated with Chromeleon/Peaknet 6.40 chromatography software, by comparing peak areas with those of known standards. Total N was determined using an automatic Kjeldahl distiller (Selecta Dosi-Gen S-511). Total S and P were measured by inductively-coupled plasma spectrometry (IRIS Intrepid II XDL, Thermo Electron Corporation, Franklin, Massachusetts) after a nitric acid (HNO3 )-perchloric acid (HClO4 ) (2:1) digestion.

Nitrate Reductase Nitrate reductase (NR, EC 1.6.6.1) was extracted and measured according to Alcaraz et al. (1979) with some modifications. Discs of young leaf tissue (100 mg) were incubated in darkness for 3 h at 27◦ C, in a reaction mixture containing 1 mL of 100 mM phosphate buffer, 2 mL of 50 mM potassium nitrate (KNO3 ),

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and 3 mL of deionized water. Then, the reaction was stopped by addition of 1 mL of 1% sulphonamide in 1.5 N hydrochloric acid (HCl) and 1 mL of N-(1naphthyl) ethylenediamine (in this order) to 3 mL of the leaf extract. The A540 of the nitrite (NO2 ) product was measured with a spectrophotometer (Spectronic Helios Alpha UV-VIS, Thermo, Massachusetts). Nitrate reductase activ−1 −1 ity was expressed as mmol NO− h . All the NR activity measurements 2 g fw were made in the middle of the photoperiod because it has been established that the activity depends on the photoperiod (Carillo et al., 2005; Tischner, 2000).

Acid Phosphatise The activity of leaf acid phosphatase (AP, EC 3.1.3.2) was determined using a method based on that described by Besford (1979). Young leaf tissue (0.5 g) was homogenised in cold conditions with 5 mL of 0.2 M acetate buffer (pH 5.8), in a pestle and mortar, and then centrifuged at 9000g for 15 min, at 4◦ C (Universal 32R, Hettich Zentrifugen, Tuttlingen). The supernatant was used as the enzyme extract. The assay mixture was 1 mL of 20 µM p-nitrophenyl phosphate with 50 µM sodium acetate buffer (pH 5.8), 0.5 mL of enzyme extract, and 0.5 mL of deionized water. This mixture was incubated for 15 min at 30◦ C. Then, the reaction was stopped with 8 mL of 0.2N NaOH. The absorbance was then read in a spectrophotometer at 405 nm (Spectronic Helios Alpha UV-VIS, Thermo, Massachusetts).

O-Acetyl-L-serine (thiol) Lyase The activity of O-Acetyl-L-serine (thiol) lyase (OASTL, EC 2.5.1.47) was measured according to the method described by Pieniaze et al. (1973) with some changes. Young leaves (1 g) were homogenized with 10 mL of 0.2 M potassium phosphate buffer (pH 8) containing 10 mM 2-mercaptoethanol and 0.5 mM ethylenediaminetetraacetic acid (EDTA). The homogenate was filtered through one layer of miracloth. The filtered extract was used for OASTL determination. The assay mixture contained, in a final volume of 200 µl: 0.2 M Tris-HCl (pH 7.5), 0.01 M DTE, 7.8 mM O-acetyl-L-serine-HCl, 7.6 mM Na2 S and an appropriate amount of extract. This mixture was incubated for 30 min at 25◦ C. The reaction was stopped by putting the glass tubes on ice and adding 125 µL of acid ninhydrin reagent (Gaitonde, 1967). The mixture was heated at 100◦ C for 30 min and then cooled rapidly on ice. Five millilitres of 99.5% ethanol were added and the absorbance was determined at 560 nm. The calibration curve was established by adding known amounts of L-cysteine to the assay mixture and measuring without incubation.

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Data Analysis The data were analysed statistically, using the SPSS 7.5 software package, by ANOVA, Tukey’s Multiple Range Test, to determine differences between means, and the CORR procedure, for the correlation analysis.

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RESULTS AND DISCUSSION Determination of fresh weight showed a decrease in salt-treated plants compared to the control and the decrease was progressive as the salinity level increased (Table 1). However, the percentage moisture content did not reveal significant differences between salt-treated and control plants. The plant weight results indicate that broccoli plants are relatively tolerant to salinity. In a previous report, we observed that broccoli plants showed a biphasic response: a greater decrease in growth in proportion to the level of salinity after one week but a lower decrease after two weeks (L´opez-Berenguer et al., 2006). Therefore, the relative growth rate in these experiments could have revealed that the plants had already adjusted their metabolism to salinity with only a slight decrease of growth. Nitrate reductase activity decreased strongly and significantly with increasing NaCl concentration in the nutrient solution from 60 to 100 mM NaCl (Figure 1). No NR activity was detected at 80 and 100 mM NaCl. At ≤ 40 mM salt levels, no significant differences were observed. Nitrate content in leaves did not differ significantly between the control and the treatments with 20, 80, or 100 mM NaCl. An increase was observed with 40 and 60 mM NaCl. In roots, nitrate values were maintained at 20 mM NaCl, but an increased was observed from 40 to 100 mM NaCl with no significant differences between these Table 1 Effect of NaCl salt applied to nutrient solution on fresh weight (g) and moisture content (%) of aerial part of broccoli plants. The values are means ± SE (n = 4). Values followed with the same letter in the same column are not significantly different (P < 0.05, Tukey test). NaCl concentration in nutrient solution mM 0 20 40 60 80 100

Fresh weight g

RHaerialpart %

151.87 ± 10.77b 152.09 ± 10.07b 149.17 ± 12.65ab 141.16 ± 11.63ab 130.81 ± 6.95a 128.73 ± 8.09a

90.24 ± 0.32 89.49 ± 0.07 90.08 ± 0.29 89.99 ± 0.06 89.25 ± 0.18 90.29 ± 0.19

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Figure 1. Effect of NaCl salt applied to nutrient solution on nitrate reductase activity in leaves and nitrate and total N content in roots and leaves. The values are means ± SE (n = 5). Columns designated with the same letter within the same tissue are not significantly different (P < 0.05, Tukey test). Statistical analysis was done separately for roots and leaves.

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Table 2 Trend analysis of nitrate, phosphate and sulphate concentration after the application of salt treatments (20, 40, 60, 80, 100 mM NaCl) for two weeks. The regression coefficient (R2 ) fitted to a quadratic equation

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NO− 3 PO3− 4 SO2− 4

Leaves Roots Leaves Roots Leaves Roots

Trend equation (R2 )

Maximum

2 ∗∗∗ [NO− ) 3 ] = −1.3256x + 15.185x + 29.349 (0.6432 − 2 [NO3 ] = −0.7773x + 11.673x + 3.3048 (0.5004∗∗∗ ) 2 ∗∗∗ [PO3− ) 4 ] = −0.1843x + 2.5423x + 4.0403 (0.4795 2 ∗∗∗ [PO3− ] = −0.3646x + 4.9719x – 0.722 (0.6706 ) 4 2 ∗∗∗ [SO2− ) 4 ] = −0.3554x + 4.6673x + 0.1874 (0.5935 2− 2 [SO4 ] = −0.1847x + 2.5891x + 1.6712 (0.4843∗∗∗ )

5.7 dS·m−1 7.5 dS·m−1 6.9 dS·m−1 6.9 dS·m−1 6.6 dS·m−1 7.0 dS·m−1

x: electrical conductivity (dS·m−1 ) ∗∗∗ : Significance at 0.01% level

treatments. A trend study (Table 2) indicated that nitrate concentration in leaves showed a maximum around 40 mM NaCl treatment (5.7 dS m−1 ) and in roots, around 55 mM NaCl treatment (7.5 dS m−1 ), following a quadratic equation. Total N in leaves increased with salt concentrations over 60 mM NaCl, with no significant differences between 80 and 100 mM, whereas in roots there were no differences among treatments. The decrease NR activity in broccoli leaves is in agreement with other studies on Zea mays, Lycopersicon esculentum, Triticum aestivum, and Bruguiera parviflora (Abd-El Baki et al., 2000; Carillo et al., 2005; Flores et al., 2000; Parida and Das, 2004; Silveira et al., 2001). Leaf NR activity seemed to increase with increasing NaCl concentration up to 40 mM. On the other hand, nitrate concentration in leaves and roots also increased with increasing NaCl concentration up to 40 mM. These results confirm that NR activity is induced by nitrate and that a lower nitrate uptake in plants subjected to salt causes an important decrease in NR activity in leaves (Akhtar et al., 2003; Bayuelo-Jimenez et al., 2003; Gebauer et al., 2003; Loupassaki et al., 2002; Netondo et al., 2004; Walker and Douglas, 1983). The primary cause of the reduction of NR activity in the leaves is a specific effect associated with the presence of Cl− salts in the external medium (Parida and Das, 2005). It has been suggested that chloride causes an inhibition of nitrate loading into the root xylem; alternatively, nitrate and chloride could compete for uptake at the plasmalemma or tonoplast of leaf cells (Carillo et al., 2005). So, chloride seems to affects NR activity by reducing nitrate uptake and, consequently, by lowering nitrate concentration in the leaves, although the possibility of a direct effect of chloride on the activity of the enzyme exists (Zekri and Parsons, 1992). Some authors suggested that the salinity-induced decrease in NR activity is not due to direct inhibitory effects of salt ions on NR activity and may instead be due to a lower uptake of nitrate (Parida and Das, 2004). Abd-El Baki et al. (2000)

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suggested that, as nitrate and chloride were sequestered in the vacuole such that concentrations in the cytosol were much lower than bulk nitrate concentrations, in salt-stress conditions nitrate loading into the xylem may depend on cytosolic nitrate concentrations in the xylem parenchyma. In previous work, we observed that chloride levels increased progressively in leaves and roots as external salinity increased (L´opez-Berenguer et al., 2006). Thus, it could be that, in conditions of low external NaCl concentration that the broccoli plants were able to tolerate, the chloride did not compete with nitrate and consequently did not affect nitrate flux. But at higher NaCl concentration that broccoli plants were not able to tolerate, chloride accumulation seriously affected nitrate content, mainly in leaves, and therefore affected NR activity. Although, it has to be taken into account that post-transcriptional processes are also important in determining protein amounts and, therefore, the activity (Carillo et al., 2005; Tischner, 2000). However, the fact that there was an increase of nitrate content in roots suggests that nitrate uptake was not inhibited and that the effect must have been on NR activity. Acid phosphatase activity was higher in salt-treated plants than in control plants (Figure 2) with positive correlation between NaCl concentration and AP activity. Leaf phosphate content showed significant decreases at 80 and 100 mM NaCl only. However, in roots, the phosphate content increased with increasing salt concentration up to 40 mM but remained at that level with further increase in salt concentration up to 100 mM. In Table 2, the trend study for phosphate (also following a quadratic equation) showed a maximum in both leaves and roots around 50 mM NaCl treatment (6.9 dS m−1 ). In leaves, total P remained constant at all treatments, whereas in roots it showed a sharp decrease as salinity increased. It has been reported that salinity causes an increase in AP with a significant decrease in phosphate level (Parida and Das, 2004). However (Szabonagy et al., 1992) also demonstrated that the induction of AP under osmotic and salt stresses was not accompanied by a decrease in phosphate level. It has been suggested that, at the whole-plant level, AP may contribute to phosphorus remobilization on an individual leaf basis (Yan et al., 2001), phosphorus remobilization from metabolically less-active mature or senescent leaves to young or developing leaves being an important determinant of phosphorus efficiency (Smith et al., 1990). The main role for leaf AP is thought to be an adaptation response to low phosphorus availability (Yan et al., 2001). Therefore, the decrease of total P in roots, together with the increase of phosphate content in roots, with salt stress could be due to the mobilisation of P from roots to leaves in order to avoid a decrease of phosphorus levels in leaves. The OASTL activity showed invariable values at different salinity levels (Figure 3). The sulfate content in leaves did not show a significant variation compared with the control, but at 40 and 60 mM NaCl the sulfate content greatly increased; similar results were detected in roots although differences were not so large. The trend study for this anion (Table 2) pointed that in leaves

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Figure 2. Effect of NaCl salt applied to nutrient solution on acid phosphatase activity in leaves and phosphate and total P content in roots and leaves. The values are means ± SE (n = 5). Columns designated with the same letter within the same tissue are not significantly different (P < 0.05, Tukey test). Statistical analysis was done separately for roots and leaves.

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Figure 3. Effect of NaCl salt applied to nutrient solution on O-acetylserine (thiol) lyase activity in leaves and sulphate and total S content in roots and leaves. The values are means ± SE (n = 5). Columns designated with the same letter within the same tissue are not significantly different (P < 0.05, Tukey test). Statistical analysis was done separately for roots and leaves.

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the maximum was around 45 mM NaCl treatment (6.6 dS m−1 ) and in roots, around 50 mM NaCl treatment (7.0 dS m−1 ). Total S, in both leaves and roots, did not show significant differences. It has been reported that exposure to different stress conditions induces higher rates of cysteine synthesis, as a result of an increased expression of the cytosolic form of OASTL (Barroso et al., 1999; Saito, 2000). Results showed OASTL does not significant change with increasing salt stress, and also there was no important variation in total S or sulfate content in leaves and roots. Other reports showed that OASTL activity increased with salt treatments, especially in salt-tolerant species (Fediuc et al., 2005). Romero et al. (2001) showed in Arabidopsis thaliana that although there was a correlation between transcript induction and cysteine level after salt treatment, there was no detectable increase of OASTL activity upon NaCl treatment. In addition, A. thaliana apparently responded to salt stress by inducing the transcription and translation of OASTL genes, probably due to a higher demand for cysteine or another sulfur-containing compound required by the plant as an adaptation or protection against higher levels of sodium (Romero et al., 2001). The role of OASTL in salt tolerance was tested in yeast and the expression of a plant OASTL gene conferred salt tolerance on yeast (Romero et al., 2001). It is interesting to note that OASTL activity stimulation is an essential early reaction mechanism, following the exposure of plants to salinity, which gradually decreases with time (Fediuc et al., 2005). In this work on broccoli plants, OASTL activity was not related to salt tolerance; it could be that broccoli adapts to salt stress by activating another metabolic pathways. The increased sulfate content in leaves at high NaCl concentrations might favour the synthesis of other S compounds like glucosinolates (data not shown), as a result of stress. The harmful effect of salinity on growth has been described as being related to plant water relations and to toxicity and/or imbalances in plant mineral nutrition (Carvajal et al., 1999). Our data indicate that salinity slightly reduced growth, but the changes in nutrient uptake and metabolism seemed to be a mechanism to compensate for the deleterious effect of NaCl. As has been observed previously, after two weeks of salinity stress, the broccoli plants could have been affected by internal injury due to Na+ or Cl− , since osmotic adjustment was achieved and water relations were relatively re-established (L´opez-Berenguer et al., 2006). However, from 60 to 100 mM NaCl, the marked responses in the nitrate, phosphate, and sulfate contents, together with the changes in the measured enzyme activities, seemed to pass the threshold of resistance.

ACKNOWLEDGMENTS The authors wish to thank Dr. D Walker for correction of the written English in the manuscript. C. L´opez-Berenguer was funded by a grant from Comunidad Aut´onoma de la Regi´on de Murcia (Spain). This work was funded by CICYT AGL2006-06499/AGR).

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