Salt-induced changes in the growth, key

Journal of Horticultural Science & Biotechnology (2013) 88 (2) 231–241

Salt-induced changes in the growth, key physicochemical and biochemical parameters, enzyme activities, and levels of nonenzymatic anti-oxidants in cauliflower (Brassica oleracea L.) By A. BATOOL1, M. ASHRAF1,2, N. A. AKRAM3* and F. AL-QURAINY2 Department of Botany, University of Agriculture, Jail Road, Faisalabad 38040, Pakistan 2 Department of Botany and Microbiology, King Saud University, Riyadh 11451, Saudi Arabia 3 Department of Botany, Government College University, Jhang Road, Faisalabad 38040, Pakistan (e-mail: [email protected]) (Accepted 5 November 2012)

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SUMMARY Salt-induced changes in the growth, leaf chlorophyll contents, water relations, inorganic nutrient contents, activities of some anti-oxidant enzymes, and levels of non-enzymatic anti-oxidants in cauliflower (Brassica oleracea L.) were examined under greenhouse conditions. Fifteen-day-old seedlings of two cultivars of cauliflower (‘FD1’ and ‘FD2’) were exposed to four different levels of saline stress [0 (control), 50, 100, or 150 mM NaCl] for 30 d, applied through the rooting medium. Increasing levels of NaCl significantly decreased shoot and root dry weights (DWs), relative water contents (RWC), leaf and root K+ ion contents, total soluble proteins, total phenolic compounds, malondialdehyde (MDA) levels, and root K+/Na+ and Ca2+/Na+ ratios. Increasing salinity increased leaf water contents, leaf and root Na+ and Cl– ion contents, leaf free proline, glycinebetaine (GB), and ascorbic acid (AsA) contents, as well as the activities of superoxide dismutase (SOD), peroxidase (POX), and catalase (CAT) in both cauliflower cultivars. However, no significant effects of NaCl were observed on chlorophyll a and b contents, relative membrane permeability (RMP), and leaf or root Ca2+ ion contents in either cauliflower cultivar. ‘FD2’ was relatively less-affected by salt stress. The relatively improved growth performance of ‘FD2’ under saline conditions was found to be attributable to its higher RWC, as well as increased levels of proline, root Ca2+ ions, total phenolic compounds and AsA, as well as elevated SOD activity, and lower levels of MDA compared to ‘FD1’ plants under salt stress.

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alt stress is known to induce several morphophysiological and metabolic disorders in photosynthesis (Noreen et al., 2010a,b; Akram and Ashraf, 2011a), nitrogen assimilation, protein synthesis, and respiration (De Pascale et al., 2005; Colla et al., 2010), nutrient uptake and transport (Akram and Ashraf, 2011b), and in the production of anti-oxidants (Ashraf, 2009; Noreen et al., 2010b) and phytohormones (Ashraf et al., 2011). All these physiological disorders result in reduced plant growth (Ashraf et al., 2010) and crop yield (Shahbaz et al., 2008; Siddiqi et al., 2011; Sabir et al., 2011). However, plant responses to salt stress are complex (Flowers et al., 2010) because they depend on a number of interconnected metabolic pathways taking place at the whole plant, tissue, and cellular levels, many of which remain to be elucidated completely (Noreen et al., 2010a, b). Up-regulation of the activities of several key antioxidant enzymes is one of the most important responses of a plant to saline stress (Ashraf, 2009). To mitigate the effects of salt stress, plants have evolved a number of antioxidant systems to protect themselves against potentially cytotoxic levels of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide radicals (O2•–), and hydroxyl radicals (•OH; Ashraf, 2009; Siddiqi et al., 2011). Various investigations have shown that plants with high levels of anti-oxidants (induced or constitutively expressed) have an increased ability to scavenge ROS and to overcome stress-induced damage (Shahbaz et al., 2008; *Author for correspondence.

Noreen et al., 2010a; Perveen et al., 2011; Siddiqi et al., 2011). However, the activities of anti-oxidant enzymes and the levels of non-enzymatic anti-oxidants differ between plant species (Sabir et al., 2011; Siddiqi et al., 2011). Plants accumulate proline (Pro) and glycinebetaine (GB) in order to mitigate adverse salt-induced effects (Chen and Murata, 2011; Shahbaz and Zia, 2011). These compounds may be involved in the maintenance of protein structure, membrane integrity, osmotic adjustment, and/or free radical-scavenging. A number of earlier reports have shown accelerated synthesis of Pro and GB in different plants under salt stress (Ashraf and Foolad, 2007; Nawaz et al., 2010; Siddiqi et al., 2011). Glycinebetaine and Pro play vital roles in the osmotic adjustment of plant cells exposed to salt stress conditions, as observed in a number of crops such as foxtail millet (Setaria italica; Veeranagamallaiah et al., 2007) and mung bean (Vigna radiata; Misra and Gupta, 2005). In general, plants with high levels of accumulation of these two osmolytes (Pro + GB) are considered to be stress tolerant, but this is not always true (Ashraf and Foolad, 2007). Increased consumption of cruciferous vegetables is particularly beneficial to human health, due to their high concentrations of essential nutrients as well as natural anti-oxidants (Podsedek, 2007; Saleem et al., 2011; Shahbaz et al., 2012). Cauliflower (Brassica oleracea L.) is an important cruciferous vegetable (Scalzo et al., 2007) because of its high contents of isothiocyanates, flavonoids, glucosinolates (Kirsh et al., 2007), and related components (Wargovich, 2000). It is rich in vitamin C,

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Salt-induced changes in cauliflower

proteins, cellulose, hemicellulose, H2O, dietary fibre, and folate, while also being low in fat (Wadhwa et al., 2006). In general, cauliflower is ranked as being moderately salt tolerant (De Pascale et al., 2005). During an earlier investigation on cauliflower, De Pascale et al. (2005) reported that increasing salinity treatments (EC 2.3 15.7 dS m–1) caused considerable reductions in marketable yields [by 9.6 – 26.9 metric tonnes (MT) ha–1] and leaf area (55%). The threshold electrical conductivity of soil extracts (ECe) was 1.52 dS m–1, and the reduction in relative yield per unit increase in ECe above this threshold was 14.4% for cauliflower (De Pascale et al., 2005). Salinity perturbs the growth of plants mainly by causing nutritional imbalances and accelerating oxidative stress. Accordingly, we hypothesised that different saline regimes could modulate the growth of cauliflower plants by altering their inorganic nutrient status, their production of enzymatic and non-enzymatic anti-oxidants, osmo-protectants and chlorophyll pigments, and their water status. The main objective of the present work was therefore to investigate saltinduced changes in the growth, leaf chlorophyll contents, water relations, inorganic nutrient levels, activities of some key anti-oxidant enzymes, and levels of nonenzymatic anti-oxidants in cauliflower.

MATERIALS AND METHODS The effects of varying levels of NaCl on cauliflower (B. oleracea) were examined at the vegetative stage, in sand culture, under greenhouse conditions from November 2010 – April 2011. Seeds of two cauliflower cultivars (‘FD1’ and ‘FD2’) were obtained from the Ayub Agricultural Research Institute, Faisalabad, Pakistan. Before the start of the experiment, the river sand was washed thoroughly, first with tap water, then with distilled water, to remove any salts previously present. The washed sand was then added to 32 plastic pots, and each pot (23.5 cm diameter 29 cm deep) contained 10 kg dry sand. A completely randomised experimental design, with four replicate pots of each cultivar and each level of NaCl was established in a greenhouse at the Botanical Garden of the University of Agriculture, Faisalabad, Pakistan. During the experiments, average (day/night) temperatures of 17.3ºC/10ºC, and an average relative humidity of 63.3% were recorded. Nutrient solution (2 l of full-strength Hoagland’s nutrient solution) was applied to the sand in each pot. Ten seeds of each cultivar were then sown directly in the moist sand in each pot (16 pots per cultivar). After 10 d, the plants were thinned to five per pot. Four levels of NaCl (0, 50, 100, or 150 mM) in Hoagland’s nutrient solution were applied to the 3week-old plants of either cauliflower cultivar in each pot. To minimise salt-shock, the NaCl concentration was raised step-wise in daily increments of 50 mM until the appropriate salt level was attained. Two litres of each treatment solution were then applied to each pot once a week for 4 weeks. Distilled H2O (250 ml) was added to each pot each day to support normal plant growth, in addition to the Hoagland’s solution with or without NaCl. After 30 d of salt treatment, two plants from each pot

were carefully uprooted, and their shoots and roots were separated. The shoots and roots were oven-dried at 65ºC for 7 d, and their dry weights (DWs) were recorded. After harvesting plants for DW measurements, the three remaining plants in each pot were used to record data for the following parameters. Chlorophyll contents Chlorophyll a and b contents were determined using the method described by Arnon (1949). Fresh leaves (0.5 g) of each plant were extracted for 12 h with 80% (v/v) acetone at –10°C. The extract was centrifuged at 14,000 g for 5 min and the absorbance of the supernatant was read at 645 nm and at 663 nm using a spectrophotometer (U2020; IRMECO, Geesthacht, Germany). Chlorophyll concentrations were determined using the formulae proposed by Arnon (1949). Water relations parameters One fully-expanded young leaf from each plant was excised and its water potential (w) was determined from 06.00 – 08.00 h using a Scholander-type pressure chamber. The same leaf used for w measurements was frozen at –20ºC for 7 d, after which time the cell sap was extracted soon after thawing. After centrifugation, the sap was used to determine the leaf osmotic potential (s) using an Vapro-type osmometer (Vapro 5500; Wescor, Logan, UT, USA). Leaf turgor potential (p) was calculated as the difference between s and w. Relative water content (RWC) One fresh leaf of uniform size from each replicate pot was used to determine RWC values following the method of Jones and Turner (1978), using the formula: Leaf fresh weight – Leaf dry weight RWC (%) = ———————————————— 100 Leaf turgid weight – Leaf dry weight Relative membrane permeability (RMP) The RMP of leaf cells was determined by the extent of ion leakage, following the method of Yang et al. (1996). Mineral nutrient concentrations Dried, ground leaf or root material (0.1 g) was placed in a digestion flask and 2 ml of a digestion mixture [i.e., 0.42 g selenium (Se) plus 14 g LiSO4.2H2O dissolved in 350 ml of H2O2, mixed well, then 420 ml conc. H2SO4 added slowly, keeping it in an ice bath] were added to each flask (Allen et al., 1985) which was then heated on a hot plate. The temperature was increased gradually from 50ºC to 200ºC. After heating the mixture at 200ºC, when it turned black, 0.5 ml of 72% (v/v) HClO4 was added to each sample, and the flask was heated again until the contents became colourless. The flasks were then removed from the hot plate and cooled. The solution was diluted to 50 ml in a volumetric flask and filtered. The filtrate was used for determinations of Na+, K+, and Ca2+ ions using a flame photometer (PFP-7; Jenway Ltd, Dunmow, UK) and standard curves based on standards of known concentrations. To determine the Cl– ion content of each plant sample, ground leaf or root material (100 mg) was extracted in 10 ml distilled water at 80ºC for 4 h. Cl– ion contents were then determined using a chloride analyser (Model 926;

A. BATOOL, M. ASHRAF, N. A. AKRAM and F. AL-QURAINY Sherwood Scientific Ltd., Cambridge, UK) and standard curves based on standards of known concentrations. Leaf free proline (Pro) contents Proline concentrations were determined following Bates et al. (1973). Each fresh leaf tissue sample (0.5 g) was homogenised in 10 ml 3% (w/v) sulpho-salicylic acid and filtered. Two ml of the filtrate was then mixed with 2.0 ml of acid ninhydrin reagent (1.25 g ninhydrin in 30 ml glacial acetic acid plus 20 ml 6 M orthophosphoric acid) and 2.0 ml of glacial acetic acid. The mixture was incubated at 100ºC for 60 min, then cooled in an ice bath. Four ml of toluene was added to the solution which was mixed well for 1 – 2 min using a stream of air. The upper (aqueous) layer was discarded and the absorbance of the lower chromophore-containing toluene layer was read spectrophotometrically at 520 nm. Proline concentrations were determined according to Bates et al. (1973). Leaf glycinebetaine (GB) contents Glycinebetaine concentrations in leaf tissues were determined following Grieve and Grattan (1983). Fresh leaf material (1.0 g) was shaken for 5 min in 10 ml of 0.5% (v/v) toluene and filtered. After filtration, 1 ml of each extract was mixed with 1 ml of 2 M H2SO4. A 0.5 ml aliquot was then added to a glass-test tube and 0.2 ml of 1 M potassium tri-iodide (KI3) solution was added, followed by 2.8 ml of ice-cooled distilled water and 6 ml of 1,2-dichloroethane (cooled to –10ºC). The upper aqueous layer was discarded, and the absorbance of the lower organic layer was measured at 365 nm. Total phenolics contents Fresh leaf tissue (0.5 g) was homogenised in 10 ml of 80% acetone and centrifuged at 10,000 g for 10 min at 4ºC. One hundred µl of the supernatant was diluted with 2 ml of water plus 1 ml of Folin-Ciocalteau’s phenol reagent, then shaken vigorously. Five ml of 20% (w/v) Na2CO3 was then added and the volume made up to 10 m with dH2O. The absorbance was read at 750 nm and the results were expressed as mg g–1 FW of leaf (Julkenen-Titto, 1985) by comparison with standards of known concentrations. Malondialdehyde (MDA) contents MDA concentrations were estimated following Carmak and Horst (1991). Each sample of fresh leaf tissue (1.0 g) was ground in 5 ml of 1.0% (w/v) trichloroacetic acid (TCA) on ice and centrifuged at 15,000 g for 10 min. Three ml of a mixture containing 0.5% (v/v) thiobarbituric acid (TBA) in 20% (w/v) TCA was then added to 0.5 ml of the supernatant. This mixture was incubated in a shaking water bath at 95ºC for 50 min. The reaction was stopped by placing the test tubes on ice and absorbance values were measured at 532 nm and at 600 nm. Ascorbic acid (AsA) contents Ascorbic acid concentrations were measured as described by Mukherjee and Choudhuri (1983). Each sample of fresh leaf material (0.25 g) was extracted with 10 ml of 6% (w/v) TCA. Four ml of each extract were then mixed with 2 ml of 2% (v/v) dinitrophenyl hydrazine in 9 M H2SO4) followed by the addition of one drop of

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10% (w/v) thiourea. The mixture was boiled for 15 min in a water bath and, after cooling, 5 ml of 80% (v/v) H2SO4 was added. The absorbance was read at 530 nm. Anti-oxidant enzyme activities Each sample of fresh leaf material (0.5 g) was ground using a pre-chilled mortar and pestle, then 5 ml of precooled 50 mM sodium phosphate buffer, pH 7.8, was added. The homogenate was vortexed and centrifuged at 15,000 g for 15 min at 4ºC. The supernatant was separated and used to assay for several key anti-oxidant enzyme activities. Superoxide dismutase (SOD) activity was determined following Giannopolitis and Ries (1977) by measuring the rate of photo-reduction of nitroblue tetrazolium (NBT). Each 3 ml reaction mixture contained 1.3 µM riboflavin, 50 µM NBT, 75 µM EDTA, 13 mM methionine, 20 mM phosphate buffer, and the 50 µl of crude leaf extract . The reaction solution was irradiated for 15 min under white fluorescent light at 80 µmol m–2 s–1. The absorbance of the solution was then read at 560 nm using an IRMECO U2020 spectrophotometer. One Unit of SOD activity was defined as the amount required to inhibit the photo-reduction of NBT by 50%. The protocol of Chance and Maehly (1955) was used to measure peroxidase (POX) and catalase (CAT) activities. Three ml of POX reaction solution was mixed with 0. 1 ml of crude leaf extract, then 0.9 ml of 40 mM H2O2, 1 ml of 20 mM guaiacol, and 1 ml of 50 mM phosphate buffer pH 5.0 were added. The change in absorbance was recorded at 470 nm every 30 s. A change in absorbance of 1.0 A470 unit min–1 was considered equal to 1 Unit of POX activity. Three ml of CAT reaction solution containing 5.9 mM H2O2 and 50 mM phosphate buffer, pH 7.8 was used to determine CAT activity. Each reaction was initiated by adding 0.1 ml of crude leaf extract. The change in absorbance at 240 nm was recorded every 20 s. A change of 0.01 A240 units min–1 was considered to be equal to 1 Unit of CAT activity. SOD, POX, and CAT activities (Units) were then expressed mg–1 total soluble protein (TSP). Protein concentrations of the crude leaf extracts were measured following Bradford (1976). Experimental design and statistical analysis A completely randomised design (CRD) was used, incorporating four levels of NaCl stress treatment and two cauliflower cultivars (‘FD1’ and ‘FD2’), each with four replications (i.e., 32 pots). The data obtained were analysed statistically by ANOVA using COSTAT Version 6.303 statistical software (Cohort Software, Monterey, CA, USA).

RESULTS Varying levels of NaCl (0, 50, 100, or 150 mM) significantly (P ≤ 0.001) decreased the DWs of shoots and roots in both cauliflower cultivars (‘FD1’ and ‘FD2’). ‘FD2’ was relatively more salt-tolerant than ‘FD1’ under all saline regimes (Table I; Figure 1A,B). No significant effect of saline level was observed on chlorophyll a and b contents, or on chlorophyll a/b ratios (Table I; Figure 1 E,F,G).

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TABLE I Analyses of variance (mean squares) in the data for different biophysical and biochemical parameters in two cauliflower (Brassica oleracea L.) cultivars (‘FD1’ and ‘FD2’) grown under saline (50, 100, or 150 mM NaCl) or non-saline conditions Parameter (units) df Shoot dry weight (g) Root dry weight (g) Chlorophyll a (mg g–1 FW) Chlorophyll b (mg g–1 FW) Leaf water potential (–MPa) Leaf osmotic potential (–MPa) Leaf turgor potential (MPa) Relative water content (%) Free proline (µg g–1 FW) Leaf GB (µg g–1 FW) Relative membrane permeability (%) Leaf Na+ (mg g–1 DW) Root Na+ (mg g–1 DW) Leaf K+ (mg g–1 DW) Root K+ (mg g–1 DW) Leaf Ca2+ (mg g–1 DW) Root Ca2+ (mg g–1 DW) Leaf Cl– (mg g–1 DW) Root Cl– (mg g–1 DW) Total phenolics (mg g–1 FW) MDA (nmol g–1 FW) H2O2 (µmol g–1 FW) Ascorbic acid (µg g–1 FW) Total soluble proteins (mg g–1 FW) SOD (Units mg–1 protein) POX (Units mg–1 protein) CAT (Units mg–1 protein) †

Cultivar (cv.) 1 1.894**† 0.313** 0.003ns 0.0004ns 0.041ns 0.004ns 0.002ns 43.95* 2048*** 763.5*** 22.13ns 0.78ns 2.53ns 51.25ns 7.51ns 0.382ns 136.1** 236.3ns 0.252ns 0.054ns 11.44ns 486.9** 101.2*** 10656.3*** 579.6*** 4.74ns 6.173ns

Salt stress (S) 3 5.64*** 0.68*** 0.008ns 0.025ns 0.035* 0.94*** 0.767*** 90.86*** 213.3*** 674.4*** 5.74ns 97.66*** 36.26*** 285.8** 34.34* 0.32ns 11.42ns 1523.3*** 72.33*** 1.844ns 7.197ns 233.1* 5.77ns 777.6* 13.78ns 1714.0ns 40.61ns

cv. S 3 0.454ns 0.214*** 0.003ns 0.006ns 0.001ns 0.019ns 0.004ns 15.17ns 0.00008ns 106.6ns 2.213ns 0.781ns 0.885ns 12.15ns 3.57ns 2.01ns 4.52ns 10.22ns 1.18ns 0.431ns 1.319ns 32.61ns 0.93ns 371.9ns 53.33ns 410.1ns 13.14ns

Error 24 0.191 0.027 0.005 0.011 0.011 0.016 0.018 9.703 1.666 53.58 7.52 7.614 2.395 41.55 9.75 8.83 16.25 83.82 2.935 1.119 2.728 54.84 7.023 211.1 30.74 596.2 45.25

ns, non-significant; *, ** and ***, significant at P ≤ 0.05, P ≤ 0.01, or P ≤ 0.001, respectively.

The leaf water potentials and leaf osmotic potentials of both cultivars decreased significantly (P ≤ 0.001) under all saline regimes (i.e., became more negative). However, leaf turgor potentials p increased in both cauliflower cultivars when the level of salt in the nutrient medium increased. The maximum increase in p was observed at 150 mM NaCl (Figure 1C,D,H). The responses of both cauliflower cultivars, regarding all the above-mentioned water relation parameters, were almost the same under all salt regimes. Salt stress (50, 100, or 150 mM NaCl) reduced the relative water content (RWC) of both cauliflower cultivars only slightly, particularly 150 mM NaCl (Table I; Figure 2A). ‘FD2’ had higher RWC values than ‘FD1’ under each saline regime. Salt stress caused a marked increase in GB concentration in both cauliflower cultivars; however, levels of GB were almost the same in both cultivars under each saline regime. GB concentrations increased more in salt-stressed ‘FD1’ plants than ‘FD2’ plants (Table I; Figure 2B). However, the latter cultivar had higher overall leaf GB concentrations than the former cultivar. Imposing varying levels of salt stress significantly (P ≤ 0.001) increased leaf Pro concentrations in both cultivars (Figure 2E); however, the response of each cultivar to salt stress varied with respect to leaf Pro concentration. ‘FD1’ had lower free proline contents than ‘FD2’ under all saline conditions. Relative membrane permeability (RMP) values remained unchanged in both cultivars under the different saline regimes. Both cultivars showed a similar response to salt stress with respect to RMP (Table I; Figure 2F). Leaf and root Na+ ion concentrations increased significantly (P ≤ 0.001) in both cultivars under all salt levels. The maximum increase in Na+ ions was observed

at 150 mM NaCl. Both cauliflower cultivars showed similar responses to salt stress with respect to leaf or root Na+ ion concentrations (Table I; Figure 2C,G). Considerable reductions in leaf and root K+ ion concentrations were observed under all saline conditions. The maximum reduction in leaf or root K+ ions was observed at 150 mM NaCl in both cultivars.The responses of both cultivars to saline stress was therefore similar with respect to these attributes (Table I; Figure 2D,H). Leaf and root Ca2+ ion concentrations were not affected by varying levels of salt stress. Both cauliflower cultivars had similar leaf Ca2+ concentrations, while ‘FD2’ had much higher root Ca2+ concentrations than ‘FD1’ (Table I; Figure 3A,E). Leaf and root Cl– ion concentrations increased significantly (P ≤ 0.001) in both cultivars under saline conditions. The responses of both cauliflower cultivars to saline stress were similar with respect to leaf or root Cl– ion concentrations (Figure 3B,F). Root K+/Na+ and Ca2+/Na+ ratios decreased significantly in both cultivars under increasingly saline conditions. The responses of both cauliflower cultivars to saline stress were similar with respect to their root K+/Na+ and Ca2+/Na+ ratios (Figure 3C,G). Ascorbic acid (AsA) concentrations in the leaves of both cauliflower cultivars increased markedly under saline conditions. The maximum increase in AsA concentration in both cultivars was observed at 150 mM NaCl. ‘FD2’ had considerably higher AsA concentrations than ‘FD1’ under saline and non-saline conditions (Table I; Figure 3H). In this study, increasing salt stress reduced the concentrations of total phenolics compounds in both cauliflower cultivars. However, ‘FD2’ was superior to ‘FD1’ in total phenolics concentrations under saline conditions (Table I; Figure 4A). Leaf MDA concentrations decreased consistently in

A. BATOOL, M. ASHRAF, N. A. AKRAM and F. AL-QURAINY

50 M NaCl

100 M NaCl

150 M NaCl

Leaf turgor potential (MPa)

Leaf osmotic potential (–MPa)

Chlorophyll a/b ratio

Leaf water potential (–MPa)

Chlorophyll b (mg g–1 FW)

Root dry weight (g plant–1)

Chlorophyll a (mg g–1 FW)

Shoot dry weight (g plant–1)

0 M NaCl

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‘FD 1’

‘FD 2’

‘FD 1’

‘FD 2’

FIG. 1 Shoot and root dry weights (Panels A,B), chlorophyll a and b contents (Panels E,F) and leaf water, osmotic and turgor potentials (Panels C,D,G,H) in two cultivars of cauliflower (Brassica oleracea L.) grown for 30 d under various levels of NaCl stress (0, 50, 100 or 150 mM). Values are means (n = 4) ± SE.

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50 M NaCl

100 M NaCl

150 M NaCl

Leaf K+ (mg g–1 DW)

Root K+ (mg g–1 DW)

Leaf Na+ (mg g–1 DW)

Root Na+ (mg g–1 DW)

Leaf glycinebetaine (µmol g–1 FW)

Relative membrane permeability (%)

Free proline (µmol g–1 FW)

Relative water content (%)

0 M NaCl

‘FD 1’

‘FD 2’

‘FD 1’

‘FD 2’

FIG. 2 Relative water content (Panel A), leaf free proline concentration (Panel E), leaf glycinebetaine concentration (Panel B), relative membrane + permeability (Panel F), leaf and root Na concentrations (Panels C,G), and leaf and root K+ concentrations (Panels D,H) in two cultivars of cauliflower (Brassica oleracea L.) grown for 30 d under various levels of NaCl stress (0, 50, 100, or 150 mM). Values are means (n = 4) ± SE.

A. BATOOL, M. ASHRAF, N. A. AKRAM and F. AL-QURAINY

50 M NaCl

100 M NaCl

150 M NaCl

Leaf H2O2 (µmol g–1 FW)

Ascorbic acid (µg g–1 FW)

Root k+/Na+ ratio

Root Ca2+/Na+ ratio

Leaf Cl– (mg g–1 DW)

Root Cl– (mg g–1 DW)

Leaf Ca2+ (mg g–1 DW)

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0 M NaCl

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‘FD 1’

‘FD 2’

‘FD 1’

‘FD 2’

FIG. 3 Leaf and root Ca2+ ion concentrations (Panels A,E), and leaf and root Cl– ion concentrations (Panels B,F), root K+/Na+ and Ca2+/Na+ ratios (Panels C,G), H2O2 concentrations (Panels D), and ascorbic acid concentrations (Panels H) in two cultivars of cauliflower (Brassica oleracea L.) grown for 30 d under various levels of NaCl (0, 50, 100, or 150 mM). Values are means (n = 4) ± SE.


Total phenolics (mg g–1 FW)

MDA (nmol g–1 FW)

SOD (Units mg–1 protein)

POX (Units mg–1 protein)

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CAT (Units mg–1 protein)

‘FD 1’

‘FD 1’

‘FD 2’

both cauliflower cultivars as the salinity of the medium increased. Overall, ‘FD1’ had higher concentrations of MDA than ‘FD2’, particularly under saline conditions. This indicates that ‘FD1’ was a relatively salt-sensitive cultivar as it exhibited higher concentrations of MDA and thus increased lipid peroxidation under NaCl stress (Table I; Figure 4B). The activities of SOD, a key anti-oxidant enzyme, remained the same under all saline regimes, in both cauliflower cultivars, although ‘FD2’ had significantly higher SOD activities than ‘FD1’ under all salt levels. No significant effects of salt regime were observed on leaf peroxidase (POX) or leaf catalase (CAT) activities in

‘FD 2’

0 M NaCl

50 M NaCl

100 M NaCl

150 M NaCl

FIG. 4 Total leaf phenolics (Panel A), malondialdehyde (MDA) concentrations (Panel B), and the activities of superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) (Panel C,D,E) in two cultivars of cauliflower (Brassica oleracea L.) grown for 30 d under various levels of NaCl stress (0, 50, 100, or 150 mM). Values are means (n = 4) ± SE.

either cauliflower cultivar. However, the patterns of POX and CAT activity were unchanged in both cauliflower cultivars under the various salinities tested (Table I; Figure 4 C,D,E).

DISCUSSION Varying levels of salt stress (50, 100, or 150 mM NaCl) significantly decreased the DWs of the shoots and roots of both cauliflower cultivars (‘FD1’ and ‘FD2’). Saltinduced reductions in the growth of glycophytic plants is well-known and has been reported by several researchers (Ashraf, 1994; 2004; Akram et al., 2008;

A. BATOOL, M. ASHRAF, N. A. AKRAM and F. AL-QURAINY Munns and Tester, 2008; Siddiqi et al., 2011; Perveen et al., 2011). In an earlier study on sunflower, Shahbaz et al. (2011) attributed the reduction in growth, measured as shoot and root FWs and DWs, to increases in leaf and root Na+ ion levels. In the present study, both cauliflower cultivars showed significant differences in shoot and root DWs, which may have been due to changes in a number of physiological and biochemical parameters linked to salt tolerance or susceptibility. For example, the accumulation of compatible solutes, increased or decreased cell membrane stability, hormonal regulation, the production of enzymatic and/or non-enzymatic antioxidants, and ion homeostasis (Ashraf, 2009; Akram and Ashraf, 2011a, b; Akram et al., 2011). In the present study, the concentrations of GB and Pro increased considerably in both cauliflower cultivars under increasingly saline regimes. In general, the accumulation of osmoprotectants such as GB and Pro under saline conditions is an important adaptation mechanism (Ashraf and Foolad, 2007; Ashraf et al., 2010). These osmoprotectants are known to play major roles in osmotic adjustments and redox homeostasis, as well as in the protection of membranes, enzymes, and proteins under osmotic stress conditions (Ashraf and Foolad, 2007; Banu et al., 2010). In the present study, the responses of both cauliflower cultivars to salt stress varied with respect to their leaf Pro and GB concentrations, as has been observed in cultivars of proso millet (Sabir et al., 2011), safflower (Siddiqi et al., 2011), cauliflower (Theriappan et al., 2011), turnip (Noreen et al., 2010a), and eggplant (Abbas et al., 2010). Measurements of the ion compositions of different plant parts (i.e., leaves, stems, and roots) can reveal the mechanism(s) of salt tolerance (Ashraf, 2004; Ulfat et al., 2007; Flowers et al., 2010). High accumulations of Na+ and Cl– ions and suppression of K+ and Ca2+ ion levels occur in most glycophytes. Such patterns of ion accumulation have recently been observed in a number of crops [e.g., okra (Saleem et al., 2011), safflower (Siddiqi et al., 2011), sunflower (Akram et al., 2009), and turnip (Noreen et al., 2010a)]. Leaf and root Na+ and Cl– ion concentrations increased considerably under our increasing saline regimes, while considerable reductions in leaf and root K+ ion concentrations were observed in both cauliflower cultivars under increasingly saline conditions. Salt-induced dehydration of cells and high Na+/K+ ratios, due to high accumulations of Na+, are known to impair various metabolic processes including plant growth and development, ion leakage, and enzyme activities (Munns and Tester, 2008), thereby resulting in reduced growth and yield, as observed in the two cauliflower cultivars used here. It is known that plant phenolic compounds act as potential anti-oxidants, and that their concentrations vary significantly depending on the sensitivity of the species to salt stress (Giorgi et al., 2009). In the present study, salt stress reduced the total phenolics concentrations of both cauliflower cultivars. However, ‘FD2’ was less affected than ‘FD1’ in terms of its phenolics concentrations under saline conditions. Noreen and Ashraf (2009b) also reported a non-significant effect of salt stress on the phenolics concentrations of radish cultivars. Furthermore, a significant increase in salt-induced total phenolics content has been reported in different plants such as pea

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(Noreen and Ashraf, 2009a), pepper (Navarro et al., 2006), and Rosmarinus officinalis (Kiarostami et al., 2010). Leaf malondialdehyde (MDA) concentrations decreased in both cauliflower cultivars under saline conditions. MDA concentration is a measure of the level of lipid peroxidation and can be used as an indicator of the extent of oxidative damage (Ashraf et al., 2010). In an earlier study on six radish cultivars, MDA concentrations were reported to decrease in some cultivars, while they increased in others under saline conditions (Noreen and Ashraf, 2009b). Furthermore, high concentrations of MDA were observed in some salt-tolerant cultivars of Panicum milliaceum, while similar levels of lipid peroxidation were recorded in some salt-sensitive accessions. This suggests that the extent of salt-induced oxidative damage depends on the species and the cultivar (Noreen and Ashraf, 2009b; Sabir et al., 2011). It has been reported that AsA acts as a non-enzymatic anti-oxidant (Dolatabadian et al., 2009). In the present study, leaf AsA concentrations increased markedly in both cauliflower cultivars under saline conditions. De Pascale et al. (2001) also observed an increase in AsA concentrations in tomato fruit grown under saline conditions. In the present study, SOD activity increased in ‘FD2’, while it decreased significantly in ‘FD1’, under saline stress. However, leaf POX and CAT activities remained largely unchanged in both cultivars under all saline conditions. Plant tolerance to salinity involves a complex network of metabolic activities, including anti-oxidant defence systems which scavenge the ROS produced under saline stress (Ashraf, 2009; Ashraf and Akram, 2009; Siddiqi et al., 2011). High accumulations of Na+ and Cl– ions damage chloroplasts and mitochondria, thereby resulting in higher accumulations of ROS, reduced photosynthetic activity, and suppressed anti-oxidant enzyme activities (Ashraf, 2009). This concurs with the findings of Noreen et al. (2010a), in which considerable reductions in the activities of SOD, POX, and CAT were observed in six turnip cultivars under various saline conditions. Overall, increasing the concentration of NaCl significantly decreased shoot and root DWs, RWC values, leaf and root K+ ion concentrations, total soluble protein levels, total phenolics concentrations, MDA concentrations, and root K+/Na+ and Ca2+/Na+ ratios. Sodium chloride also increased leaf water and leaf osmotic potentials, Na+ and Cl– ion concentrations, leaf Pro, GB, and AsA concentrations, and the activities of SOD, POX, and CAT. However, no significant effects of NaCl were observed on chlorophyll a and b concentrations, RMP, or Ca2+ ion concentrations in either cauliflower cultivar. The relatively better growth performance of ‘FD2’ under saline regimes was attributable to its higher RWC, free proline, root Ca2+ ion, total phenolics, and AsA concentrations, as well as elevated activity of SOD and lower MDA concentrations than in ‘FD1’ plants grown under salt stress. The authors acknowledge funding from the Pakistan Academy of Sciences (Grant No. 5-9/PAS/4778) and from the King Saud University, Riyadh, Saudi Arabia (Research Grant No. KSU-VPP-101).

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