Inducing Salt Tolerance in Wheat by Exogenously Applied Ascorbic Acid through Different Modes

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LPLA #424407, VOL 32, ISS 11

Inducing Salt Tolerance in Wheat by ExogenouslyApplied Ascorbic Acid through Different Modes

Habib-ur-Rehman Athar, Ameer Khan, and Muhammad Ashraf

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Journal of Plant Nutrition, 32: 1–19, 2009

Copyright © Taylor & Francis Group, LLC

ISSN: 0190-4167 print / 1532-4087 online

DOI: 10.1080/01904160903242334

Inducing Salt Tolerance in Wheat by Exogenously1

Applied Ascorbic Acid through Different Modes2

Habib-ur-Rehman Athar,1 Ameer Khan,2,3 and Muhammad Ashraf23

1Institute of Pure and Applied Biology, Bahauddin Zakariya University,4Multan, Pakistan5

2Department of Botany, University of Agriculture, Faisalabad, Pakistan63Department of Botany, University of Sargodha, Sargodha, Pakistan7

ABSTRACT8

In order to assess whether exogenous application of ascorbic acid (AsA) through dif-9ferent ways could alleviate the adverse effects of salt-induced adverse effects on two10wheat cultivars differing in salinity tolerance, plants of a salt tolerant (‘S-24’) and a11moderately salt sensitive (‘MH-97’) cultivar were grown at 0 or 120 mM sodium chlo-12ride (NaCl). Ascorbic acid (100 mg L−1) was applied through the rooting medium, or13as seed soaking or as foliar spray to non-stressed and salt stressed plants of wheat.14Salt stress-induced reduction in growth was ameliorated by exogenous application of15ascorbic acid through different ways. However, root applied AsA caused more growth16enhancement under saline conditions. Leaf ascorbic acid, catalase (CAT), peroxidase17(POD), and superoxide dismutase (SOD) activities were also maximal in salt stressed18plants of both cultivars treated with AsA through the rooting medium. Furthermore, leaf19ascorbic acid, CAT, POD, and SOD activities were higher in salt stressed plants of ‘S-24’20than those of ‘MH-97’. Root applied AsA caused more enhancements in photosynthetic21rate. Root applied AsA caused more reduction in leaf sodium (Na+) compared with AsA22applied as a seed soaking or foliar spray. Overall, AsA-induced growth improvement in23these two wheat cultivars under saline conditions was cultivar specific and seemed to24be associated with higher endogenous AsA, which triggered the antioxidant system and25enhanced photosynthetic capacity.26

Keywords: antioxidants, foliar spray, ion homeostasis, photosynthesis, salt tolerance,27seed soaking28

Received 4 October 2007; accepted 11 May 2008.Address correspondence to M. Ashraf, Department of Botany, University of Agricul-

ture, Faisalabad 38040, Pakistan. E-mail: [email protected]

1


2 H. Athar et al.

INTRODUCTION29

Salinity stress poses major challenges to crop growth and yield in various crop30species (Munns, 2005). Like other abiotic stresses, salinity stress reduces plant31growth and yield due to changes in various physiological and biochemical char-32acteristics such as reduced photosynthetic metabolism, leaf chlorophyll content33and photosynthetic capacity, diversion of energy in the processes of osmotic34adjustment and ion exclusion, and nutritional imbalance (Dubey, 2005; Munns,352005). There is an ample evidence that decline in net photosynthesis under36salt stress is primarily due to stomatal and non-stomatal limitations (Dubey,372005). In addition, it is now widely accepted that reactive oxygen species38(ROS) are generated in sub-cellular compartments such as chloroplasts and39mitochondria in response to abiotic stresses including salt stress (Mittler, 2002;40Foyer and Noctor, 2003). Stress-induced production of ROS is responsible for41various stress-induced damage to macromolecules and ultimately to cellular42structure (Smirnoff, 2005). Thus, all these salt-induced changes in biochemical43and physiological processes contribute in salt-induced-reduction in growth and44yield.45

Plants exhibit a variety of biochemical and physiological responses to46acclimatize saline environment (Munns, 2002, 2005). Of these salt-induced47changes, accumulation of compatible solutes, certain free radical scavenging48compounds and enzymes, and specific proteins that control ion and water home-49ostasis (Ashraf, 2004; Ashraf and Harris, 2004). However, enhance antioxidant50capacity of plants to scavenge reactive oxygen species (ROS) is one of the most51critical requirement. In view of a number of studies, salt tolerance is often cor-52related well with a more efficient oxidative system (Gossett et al., 1994, 1996;53Bor et al., 2003). An efficient antioxidant system comprises non-enzymatic an-54tioxidants (ascorbate, salicylate, glutathione, tochopherols etc.) and enzymatic55antioxidants like superoxide dismutase (SOD), ascorbate peroxidase (APX)56and catalase (CAT) (Foyer and Noctor, 2003). Among them, ascorbic acid57(AsA) is one of the most important antioxidants abundantly occurring in plants58(Smirnoff, 2005). In addition to its role as antioxidant, it also has a role in cell59division and cell enlargement, stomatal regulation, and floral induction (Barth60et al., 2006).61

Enhanced production of antioxidants for enhanced crop salt tolerance can62be achieved through breeding or if the internal level of such antioxidants in63a plant species is inherently low, the desired level can be achieved through64exogenous application of specific antioxidants (Ashraf and Foolad, 2007).65It has been observed that exogenous application of AsA counteracts salt-66induced growth inhibition in plants, e.g., wheat (Al-Hakimi and Hamada, 2001;67Al-Hakimi, 2001), and tomato (Shalata and Neumann, 2001). However, in view68of some earlier studies it is evident that the extent of effectiveness of exoge-69nously applied compounds depends on mode of application (Heuer, 2003). For70example, foliarly applied glycinebtaine (GB) improved salt tolerance in wheat71


Inducing Salt Tolerance in Wheat by Ascorbic Acid 3

(Raza et al., 2006), but in contrast exogenous application of GB and proline72through the rooting medium caused inhibitory effects on growth of tomato73(Heuer, 2003).74

In order to appraise the extent of effectiveness of AsA application through75different means in inducing salt tolerance in wheat, the present study was76conducted to compare the effects of exogenously applied AsA through rooting77medium, applied foliarly or as pre-sowing seed treatment on wheat plants grown78under saline conditions. It was also assessed that up to what extent exogenously79applied AsA could reverse the adverse effects of salt stress on growth, antioxi-80dant capacity, photosynthetic capacity, and ion and water homeostasis in wheat81plants.82

MATERIALS AND METHODS83

A hydroponic experiment was conducted during the winter of 2004–2005 in84a net-house at the Botanic Gardens of the University of Agriculture, Faisal-85abad, Pakistan (latitude 31◦30 N, longitude 73◦10 E and altitude 213 m), with8610/14 light/dark period at 800–1100 µmol m−2 s−1 PPFD, a day/night av-87erage temperature cycle of 26/15◦C, and 65 ± 5% relative humidity. Seeds88of a salt tolerant (‘S-24’) and a moderately salt sensitive cultivar (‘MH-97’)89of spring wheat were obtained from the Department of Botany, University of90Agriculture, Faisalabad, Pakistan and Ayub Agricultural Research Institute,91Faisalabad, Pakistan, respectively. The seeds of both cultivars were surface92sterilized with 5% sodium hypochlorite for five minutes and then thoroughly93rinsed with distilled water before further experimentation. Seeds (100 seeds of94each cultivar; 25 seeds per Petri plate) of both cultivars were allowed to germi-95nate on filter paper moistened with 0 mM or 150 mM sodium chloride (NaCl)96solutions. After seven days, wheat seedlings of both cultivars were transferred97to plastic containers (45 × 66 × 23 cm) containing 20 L 0 mM or 150 mM98NaCl solutions for further four week period. Ascorbic acid (100 mg L−1 AsA)99applied through the rooting medium, seed soaking or as a foliar spray to wheat100plants of both cultivars at vegetative stage growing in Hoagland’s nutrient so-101lution plus 0 or 150 mM NaCl. Tween-20 (0.1%) was used as a surfactant for102foliar spray. However, in the seed soaking treatment, seeds of both cultivars103were soaked in 0 (control, water), 100 mg L−1 AsA for six hours and then104air dried before further experimentation. The treatment solutions continuously105aerated and were replaced weekly. After four weeks of growth, the following106physiological biochemical attributes were measured.107

Gas Exchange Parameters108

An open system LCA-4 ADC portable infrared gas analyzer (Analytical De-109velopment Company, Hoddesdon, England) was used for the measurements of110


4 H. Athar et al.

gas exchange parameters such as net CO2 assimilation rate (A), transpiration111(E), substomatal conductance (C i), and stomatal conductance (gs). All mea-112surements were made on a fully expanded third leaf from top of each plant.113Measurements were performed from 9.00 to 13.00 hours with the following114specifications/adjustments: molar flow of air per unit leaf area 403.3 µmol115m−2s−1, atmospheric pressure 99.9 kPa, water vapor pressure into the chamber116ranged from 6.0 to 8.9 mbar, PAR at the leaf surface was maximum up to 1711117µmol m−2 s−1, temperature of the leaf ranged from 28.4 to 32.4◦C, ambient118temperature ranged from 22.4 to 27.9◦C, and ambient CO2 concentration was119352 µmol mol−1.120

Water Relations121

Leaf water potential measurements were made with a Scholander type pres-122sure chamber (Arimad-2, ELE International, Tokyo, Japan). Third leaf from123each plant was excised at 7.00 a.m. and inserted into pressure chamber for124determining leaf water potential. A proportion of the same leaf used for water125potential measurements, was frozen into 2 cm3 polypropylene tubes at −40◦C126in an ultra-low freezer for two weeks, after which time the leaf material was127thawed and the frozen sap was extracted by crushing the material with a glass128rod. After centrifugation (8000 g) for four minutes, the sap was directly used129for osmotic potential determination using a vapor pressure osmometer (Wescor1305520, Wescor Inc., Logan, UT, USA). Leaf turgor pressure was calculated as131the difference between leaf water potential and leaf osmotic potential values.132

Extraction of Antioxidant Enzymes133

Fresh leaves (0.5 g of third leaves) of both wheat cultivars were ground in1348 mL of 50 mM cold phosphate buffer (pH 7.8) and centrifuged at 15000 g for13520 min at 4◦C. The supernatant was used for the determination of the activities136of antioxidant enzymes.137

Superoxide Dismutase (SOD)138

The activity of SOD was assayed following the method of Giannopolitis and139Ries (1977) which measures its ability to inhibit the photochemical reduction140of nitroblue tetrazolium (NBT) The reaction solution (3 mL) contained 50 µM141NBT, 1.3 µM riboflavin, 13 mM methionine, 75 nM ethylenediaminetetraacetic142acid (EDTA), 50 mM phosphate buffer (pH 7.8), and 20 to 50 µL enzyme143extract. The test tubes containing the reaction solutions were irradiated under a144light (15-W fluorescent lamps) at 78 µmol m−2 s−1 for 15 min. The absorbance145



of the irradiated solution at 560 nm was determined with a spectrophotometer146(Hitachi U-2100, Tokyo, Japan). One unit of SOD activity was defined as the147amount of enzyme which caused 50% inhibition of photochemical reduction148of NBT.149

Catalase (CAT) and Peroxidase (POD)150

Activities of CAT and peroxidase (POD) were appraised following the method151of Chance and Maehly (1955) with some modification. The CAT reaction152solution (3 mL) contained 50 mM phosphate buffer (pH 7.0), 5.9 mM hydrogen153peroxide (H2O2), and 0.1 mL enzyme extract. The reaction was initiated by154adding the enzyme extract. Changes in absorbance of the reaction solution155at 240 nm were read every 20 sec. One unit CAT activity was defined as an156absorbance change of 0.01 units per min. The POD reaction solution (3 mL)157contained 50 mM phosphate buffer (pH 5.0), 20 mM guaiacol, 40 mM H2O2,158and 0.1 ml enzyme extract. Changes in absorbance of the reaction solution at159470 nm were determined every 20 sec. One unit POD activity was defined as160an absorbance change of 0.01 units per min.161

The activity of each enzyme was expressed on protein basis. Protein con-162centration of the crude extract was measured by the method of Bradford (1976).163

Ascorbic Acid164

Ascorbic acid was determined as described by Mukherjee and Choudhuri165(1983). Leaf material (0.25 g of the third leaf) was extracted with 10 mL166of 6% trichloroacetic acid. Four ml of the extract were mixed with 2 mL of 2%167dinitrophenyl hydrazine (in acidic medium) followed by the addition of one168drop of 10% thiourea (in 70% ethanol). The mixture was boiled for 15 min in169a water bath and after cooling at room temperature, 5 mL of 80% (v/v) sulfuric170acid (H2SO4) were added to the mixture at 0◦C. The absorbance was read at171530 nm. The concentration of ascorbic acid was calculated from a standard172curve plotted with known concentrations of ascorbic acid.173

Four weeks after the commencement of salt stress and AsA treatment, nine174plants from each plastic tank (three plants per replicate) were removed. Shoots175and roots were weighed for fresh weights and then all samples were oven-dried176at 65◦C for one week for recording dry weights.177

Determination of Mineral Elements in Plant Tissues178

Sodium (Na), potassium (K+), and calcium (Ca2+) in the leaves and roots179were determined by the methods described by Allen et al. (1986). Ground180


6 H. Athar et al.

dry plant samples (100 mg each) were digested in 2 mL of sulfuric-peroxide181digestion mixture until a clear and almost colorless solution was obtained.182After digestion, the volume of each sample was made to 100 mL with distilled183de-ionized water. Ions, i.e., Na+, K+, and Ca2+ were determined with a flame184photometer (Jenway PFP7; Bibby Sterilin, Essex, UK).185

Statistical Analysis of Data186

The data for each variable was subjected to analysis of variance using the187COSTAT computer package (Cohort Software, Berkeley, CA, USA). The mean188values were compared with the least significance difference test following189Snedecor and Cochran (1980).190

RESULTS191

Salt stress reduced the shoot fresh and dry weights of both wheat cultivars192(Table 1). However, exogenous application of AsA improved the shoot fresh193and dry weights of both wheat cultivars (Table 1). Since the interaction terms194(MOP × Salt; Salt × Cvs × MOP) were non-significant, it was not possible195to compare the means of each cultivar at different modes of AsA application.196

Table 1Mean squares from analyses of variance (ANOVA) of the data for shoot fresh weight,shoot dry weight, leaf ascorbic acid (AsA), superoxide dismutase (SOD), catalase (CAT),peroxidase (POD) of two spring wheat cultivars, when AsA was exogenously appliedto salinity stressed and non-stressed plants through different modes

Source of Shoot fresh Shoot dryvariation df weight weight AsA SOD CAT POD

Salt 1 2135.52∗∗∗ 83.69∗∗∗ 1594.39∗∗∗ 998.12∗∗∗ 1406.55∗∗∗ 5116.37∗∗∗

Cultivars (Cv) 1 122.32∗∗∗ 3.12∗∗∗ 314.34∗∗∗ 209.52∗∗∗ 18.17ns 129.68∗∗

Mode ofApplication(MOP)

3 43.54∗∗∗ 6.66∗∗∗ 108.86∗∗ 54.96∗∗∗ 142.79∗∗∗ 299.13∗∗∗

Salt × Cv 1 6.293ns 0.057ns 11.733ns 11.60ns 161.58∗∗ 38.79nsSalt × MOP 3 5.891ns 0.303ns 15.055ns 37.41∗∗ 49.47∗ 32.93nsCv × MOP 3 5.124ns 0.354ns 9.0564ns 14.09ns 99.84∗∗ 61.89∗

Salt × Cv ×MOP

3 10.279ns 0.133ns 13.017ns 13.29ns 14.83ns 26.42ns

Error 48 3.978 0.143 18.071 7.15 16.07 15.73Total 63

∗, ∗∗, ∗∗∗ = significant at 0.05, 0.01 and 0.001 levels, respectively; ns = non-significant.



However, it is clear from the mean values (Table 2) that root applied AsA197improved the shoot fresh dry weights of both wheat cultivars under both saline198and non-saline conditions. Moreover, AsA-induced ameliorative effect on these199growth attribute was more in salt stressed plants of cv. ‘S-24 ‘compared with200those of MH-97 (Table 2).201

Salt stress significantly reduced leaf ascorbic acid contents of both culti-202vars (Table 1). Exogenous application of ascorbic acid through different ways203caused a significant effect on leaf ascorbic acid contents (Table 1). Moreover,204root applied AsA enhanced ascorbic acid in the salt stressed leaves of both205cultivars (Figure 1). From Figure 1 it is obvious that root applied AsA caused206a greater increase in leaf ascorbic acid of salt stressed plants of ‘S-24’ than207those of ‘MH-97’. However, cultivars did not differ in leaf AsA contents under208saline conditions when AsA applied as a foliar spray or as a seed soaking209treatment. Salt stress caused a significant effect on activities of all the three210antioxidant enzyme activities (Table 1). Salt stressed plants of both cultivars211showed higher activities of superoxide dismutase (SOD), catalase (CAT), and212peroxidase (POD) than those in non-stressed plants (Figure 1). Ascorbic acid213applied only through the rooting medium significantly increased the CAT, POD214

Figure 1. Leaf ascorbic acid content, and activities of catalase (CAT), superoxidedismutase (SOD), and peroxidase (POD) of salt stressed and non-stressed plants of twospring wheat cultivars at booting stage when 100 mg L-1 ascorbicic acid was appliedthrough different ways.


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3.2

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314.

75±

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2.27

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313

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1.77

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8



Table 3Mean squares from analyses of variance (ANOVA) of the data for leaf chlorophyll a (Chla), net CO2 assimilation rate (A), transpiration rate (E), sub-stomatal CO2 (Ci), stomatalconductance (gs), and water use efficiency (WUE) of two spring wheat cultivars, whenAsA was exogenously applied to salinity stressed and non-stressed plants throughdifferent modes

Source of A/Evariation df Chl a A E Ci gs (WUE)

Salt 1 0.483∗∗∗ 390.47∗∗∗ 12.15∗∗∗ 1066.79∗∗∗ 142364.02∗∗∗ 40.16∗∗∗

Cultivars (Cv) 1 0.060∗∗∗ 10.67∗∗∗ 0.697∗ 0.088ns 8376.45∗∗∗ 0.163nsMode of

Application(MOP)

3 0.017∗ 20.72∗∗∗ 2.594∗∗∗ 174.96∗ 8722.91∗∗∗ 0.434ns

Salt × Cv 1 0.001ns 0.20ns 0.001ns 104.96ns 7491.85∗∗∗ 0.088nsSalt × MOP 3 0.001ns 3.43∗∗∗ 0.196ns 286.39∗∗ 918.99∗∗ 1.269∗∗

Cv × MOP 3 0.001ns 1.24∗∗∗ 0.836∗∗∗ 150.64ns 2407.38∗∗∗ 1.562∗∗

Salt × Cv ×MOP

3 0.002ns 0.195ns 0.079ns 203.18∗ 514.01∗ 0.409ns

Error 48 0.004 0.184 0.114 54.29 159.86 0.297


and SOD activity in the salt stressed plants of ‘S-24’, whereas those of ‘MH-97’215only CAT and POD activities were increased (Figure 1).216

A significant reduction in leaf chlorophyll a due to imposition of salt217stress was observed in both cultivars (Table 3). Both cultivars also differed218significantly in this biochemical attribute. Although exogenously applied AsA219caused a significant effect on leaf chlorophyll aof salt stressed plants of both220cultivars, this effect was slightly observed when AsA applied through the root-221ing medium (Figure 2). Saline growth medium caused a significant reduction222in all gas exchange attributes (Table 3; Figure 2). Although interactive term223salt × Cvs was non-significant, salt stressed plants of cv ‘S-24’ exhibited higher224A than that of ‘MH-97’ (Figure 2). However, cultivars did not differ in A un-225der non-saline conditions. Exogenous application of AsA through the rooting226medium caused more increase in net carbon dioxide (CO2) assimilation rate in227both cultivars under both non-saline and saline conditions (Figure 2). Although228imposition of salt stress caused a significant reduction in sub-stomatal CO2229(C i), cultivars did not differ in this physiological attribute. However, exoge-230nous application of AsA through seed soaking treatment caused increase in231sub-stomatal CO2 (C i). However, AsA applied through the rooting medium or232as a foliar spray caused an increase in stomatal conductance (gs) of both cul-233tivars under saline conditions, whereas under non-saline conditions the same234was true only for cv ‘S-24’. Salt induced-reduction in transpiration rate (E)235


10 H. Athar et al.

Figure 2. Leaf chlorophyll “a”, and gas exchange characteristics of salt stressed andnon-stressed plants of two spring wheat cultivars at booting stage when 100 mg L-1 ofascorbicic acid was applied through different ways.

of both cultivars increased due to root applied AsA or foliar applied AsA,236whereas this kind of effect of AsA was not observed due to AsA application as237seed soaking treatment. Furthermore, water use efficiency (calculated as A/E238WUE) of non-stressed plants of both cultivars was increased due to AsA ap-239plied through the rooting medium, but this was not true for the stressed plants240(Figure 2).241

Salt stress had significant adverse effects on all leaf water relation param-242eters such as water potential, osmotic potential, and turgor potential of both243cultivars (Table 4; Figure 3). Although exogenously applied AsA did not alter244leaf water potential and osmotic potential of the salinized plants of both cul-245tivars, leaf turgor potential of salinized plants of both cultivars was improved246(Figure 3).247



Table 4Mean squares from analyses of variance (ANOVA) of the data for leaf water potential,leaf osmotic potential and leaf turgor potential of two spring wheat cultivars, when AsAwas exogenously applied to salinity stressed and non-stressed plants through differentmodes

Source of variation df Water potential Osmotic potential Turgor potential

Salt 1 3.079∗∗∗ 1.924∗∗∗ 0.086∗∗

Cultivars (Cv) 1 0.101∗∗ 9.62e-7ns 0.015nsMode of Application

(MOP)3 0.014ns 0.018∗ 0.083∗∗∗

Salt × Cv 1 4.5517e-4ns 0.0027ns 0.062∗

Salt × MOP 3 0.027∗ 0.023∗ 0.029∗

Cv × MOP 3 0.0101ns 0.005ns 0.021nsSalt × Cv × MOP 3 0.0113ns 0.016∗ 0.018nsError 48 0.0097 0.005 0.008


Accumulation of Na+ in the leaves and roots of both wheat cultivars was248significantly increased under saline conditions (Table 5; Figure 4). Exogenous249application of AsA enhanced the accumulation of Na+ in both leaves and roots250of salt stressed plants of both cultivars, particularly in salt stressed plants of251‘MH-97’ due to seed soaking with AsA (Figure 4). Accumulation of K+ and252Ca2+ in the leaves and roots of both cultivars was significantly reduced due to253salt stress (Table 5; Figure 4). However, application of AsA through the rooting254medium enhanced the accumulation of K+ in the leaves and roots of the salt-255stressed plants of both cultivars. Moreover, this AsA-induced enhancement in256K+ was also observed in the roots of salt stressed plants of ‘MH-97’ due to its257foliar application. Similarly, accumulation of Ca2+ in the leaves of salinized258plants of both cultivars was also enhanced due to AsA application. However,259this effect was more pronounced in the leaves and roots of ‘S-24’ due to AsA260application as a foliar spray under saline conditions. Saline growth medium261caused a significant reduction in leaf and root K+/Na+ ratios in both wheat262cultivars. However, exogenously applied AsA through the rooting medium263caused a further reduction in leaf K+/Na+ ratio in both cultivars (Figure 4),264whereas the same was true for root K+/Na+ ratio due to AsA application as265seed soaking treatment.266

DISCUSSION267

In the present study, growth of both cultivars was improved due to exogenous268application of AsA, particularly when applied through the rooting medium.269


12 H. Athar et al.

Figure 3. Leaf water potential, osmotic potential and turgor potential of salt stressedand non-stressed plants of two spring wheat cultivars at booting stage when differentconcentrations of ascorbicic acid was applied through different ways.

These results are similar with those of in which it has already been observed270that exogenous application of AsA counteracts salt-induced growth inhibition271in plants, e.g., wheat (Al-Hakimi and Hamada, 2001; Al-Hakimi, 2001), and272tomato (Shalata and Neumann, 2001). Furthermore, differential effect of AsA273applied through different modes on growth of both wheat cultivars under nor-274mal or saline conditions can be related with some earlier studies in which it275


Tabl

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vari

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(AN

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1∗∗∗

16.2

0∗∗∗

42.7

3∗∗∗

6.56

8ns

2.49

6∗∗∗

6.63

1∗∗2.

521∗∗

∗0.

165n

sSa

lt×

Cv

114

.23∗∗

∗0.

279n

s7.

236n

s8.

353n

s0.

013n

s0.

134n

s0.

028n

s0.

211n

sSa

lt×

MO

P3

18.4

6∗∗∗

26.7

6∗∗∗

1.84

4ns

12.1

8∗∗0.

205n

s2.

692n

s0.

700n

s0.

859∗∗

Cv

×M

OP

310

.20∗∗

∗0.

375n

s5.

067n

s14

.17∗∗

2.35

0∗∗∗

10.8

2∗∗∗

1.86

7∗∗0.

738∗∗

Salt

×C

v×

MO

P3

6.37

4∗∗∗

2.04

6ns

6.06

0ns

4.64

8ns

0.91

1∗2.

101n

s2.

163∗∗

∗0.

134n

sE

rror

480.

875

1.70

35.

497

2.47

00.

229

1.22

90.

312

0.15

6

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14 H. Athar et al.

Figure 4. Na+, K+, Ca+2 concentration in the leaves and roots, and leaf and root

K+/Na+ ratios of salt stressed and non-stressed plants of two spring wheat cultivarsat booting stage when different concentrations of ascorbicic acid was applied throughdifferent ways.

was found that the extent of effectiveness of exogenously applied compatible276solutes depends on mode of application (Heuer, 2003). For example, foliarly277applied glycinebtaine counteracted growth inhibition of wheat induced by salt278(Raza et al., 2006; 2007), but in contrast exogenous application of GB and pro-279line through the rooting medium caused inhibitory effects on growth of tomato280



(Heuer, 2003). One of various possible reasons of this differential effects of281AsA applied through different ways could be the differential enhancement in282AsA-induced antioxidant capacity. For example, leaf AsA was higher in the283salt stressed plants of ‘S-24’ than that in those of MH-97 due to root applied284AsA. However, such differences in leaf AsA in these wheat cultivars could285not be seen in plants treated with AsA as a foliar spray or as a seed soaking286treatment. Similarly, CAT, POD, and SOD activities were increased due to root287applied AsA in the salt stressed plants of ‘S-24’, whereas that in ‘MH-97’288only CAT and SOD activities were increased. Secondly, salt stressed plants289of cv ‘S-24’ had higher SOD and POD activities as compared with those of290‘MH-97’ plants (Figure 1). These findings support the view of different re-291searchers that salt tolerance in most crop plants is associated with a more292efficient antioxidative system (Gossett et al., 1994; 1996; Bor et al., 2003).293Since the major enzymes involved in scavenging ROS are CAT, POD, and294SOD (Mittler, 2002; Jung, 2004), it is assumed that CAT-POD-SOD antiox-295idant system is necessary for improving salt tolerance as has been observed296in the present study in cv ‘S-24’. Although root applied AsA improved SOD297and CAT activity in salt stressed plants of ‘MH-97’, POD activity remained298unchanged under saline conditions–the second important H2O2 scavenging299enzyme. While working with wheat, Sharma et al. (2005) found higher POD Q1300activity in salt tolerant ‘K-65’ as compared to salt sensitive ‘HD2329’ under301saline conditions. Thus, higher salt tolerance in ‘S-24’ due to root applied AsA302was due to higher leaf AsA and more efficient CAT-POD-SOD antioxidant en-303zyme system. In a similar study, Zhao and Zou (2002) reported that exogenous304application of phenolics and antioxidants compounds induced abiotic stress305tolerance in wheat. This view was further supported by Shalata and Neumann306(2001) who reported that root applied AsA improved the growth of tomato307seedlings under saline conditions by specifically improving its antioxidant308capacity.309

From the results of the present study, it is amply clear that imposition of310salt stress increased the leaf Na+ coupled with a decrease in leaf Ca2+ and K+311which resulted in lower plant water status in both wheat cultivars (Figures 3312and 4), the typical osmotic and toxic effects of salt stress on plants as reported313in some extensive reviews (Ashraf, 1994, 2004; Munns, 2002, 2005). Salt314stress conditions also caused stomatal closure thereby reducing CO2 fixation,315which in turn resulted in lower biomass production under saline conditions316(Table 2; Figure 3). It is already known in the literature that salt-induced317stomatal closure not only inhibits the CO2 fixation by reducing CO2/O2 ra-318tio in the leaves, but it also causes the production of reactive oxygen species319(ROS) in the chloroplast, which cause photooxidative damage to photosyn-320thetic apparatus (Mittler, 2002; Foyer and Noctor, 2003). From the results321of the present study, it is also clear that salt stress reduces the chlorophyll322content in both wheat cultivars and exogenous application of AsA improve323photosynthetic pigments which in turn positively correlated with AsA-induced324


16 H. Athar et al.

enhanced A. Similar positive relationship between leaf chlorophyll and net CO2325assimilation rate has already been observed in sunflower (Ashraf and Sultana,3262000).327

It was also observed that root applied AsA caused an increase in photo-328synthetic rate (A) coupled with increase in stomatal conductance (gs) of the329salt stressed plants of both cultivars indicating that A was mainly controlled by330stomatal factors. This view is supported by the fact that AsA has an important331role in stomatal regulation (Chen and Gallie, 2004). However, AsA-induced332increase in substomatal CO2 (C i) of the salt stressed plants of both cultivars was333also observed when applied as a foliar spray or as a seed soaking treatment,334which indicates that changes in photosynthesis due to exogenously applied335AsA may have been due to some non-stomatal factors that are induced at lower336level of endogenous AsA. Furthermore, leaf Na+ was higher in salt stressed337plants of both cultivars when AsA applied as a foliar spray or as a seed soaking338treatment. It is already known that high accumulation of Na+ in the leaves cause339the degradation of chlorophyll in sunflower (Ashraf and Sultana, 2000), wheat340(Raza et al., 2007; Arfan et al., 2007). From these findings, it can be proposed341that root applied AsA-induced enhancement in photosynthetic pigments might342have been due to its protective effect from its degradation either through its343direct toxic effect or salt-induced oxidative stress on chlorophyll biosynthesis344resulting in increased photosynthetic capacity of wheat plants. Thus, lower345Na+ contents in the leaves of both cultivars treated with root applied AsA was346possibly one of the reasons of AsA-induced enhancement in A through some347non-stomatal factors.348

Plant water status of salt stressed plants of both cultivars was also more349improved when AsA applied through the rooting medium as compared to AsA350applied through other modes. Maintenance of plant water status is necessary351for plant growth and development (Munns, 2002). Furthermore, a positive as-352sociation has been found between plant water status and leaf ascorbic acid353or photosynthetic capacity, which indicates that differential effects of AsA354applied through different modes may have been due to differential uptake355of AsA which improves plant water status, protects the photosynthetic ap-356paratus from salt-induced oxidative stress thereby improving photosynthetic357capacity.358

Overall, only root applied AsA applied significantly alleviated the ad-359verse effects of salt stress on growth of both wheat cultivars. However, this360effect was cultivar specific. Moreover, exogenous application of AsA through361different modes caused differential uptake of AsA, which triggered CAT-POD-362SOD antioxidative system, and maintained ion and water homeostasis thereby363protecting photosynthetic machinery of wheat against salt-induced oxidative364stress. However, the detailed mechanism of differential uptake of AsA due365to exogenous application of AsA through different modes needs to be further366researched.367



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Inducing Salt Tolerance in Wheat by Exogenously Applied Ascorbic Acid through Different Modes

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