ARTICLE IN PRESS Journal of Plant Physiology ] (]]]]) ]]]—]]]

www.elsevier.de/jplph

Pretreatment of seed with H2O2 improves salt tolerance of wheat seedlings by alleviation of oxidative damage and expression of stress proteins Abdul Wahida,, Mubaraka Perveena, Sadia Gelania, Shahzad M.A. Basrab a

Department of Botany, University of Agriculture, Faisalabad-38040, Pakistan Department of Crop Physiology, University of Agriculture, Faisalabad-38040, Pakistan

b

Received 2 November 2005; accepted 11 January 2006

KEYWORDS H2O2 absorption; Membrane permeability; ROS scavenging; Signaling; Stress proteins

Summary Increased salinity is a stringent problem to crop production while seed pretreatment can effectively induce salt tolerance in plants. Hydrogen peroxide (H2O2), a stress signal molecule, was evaluated as seed treatment to produce the metabolic changes, which could lead to improved salt tolerance in wheat. Soaking in 1, 40, 80 and 120 mM H2O2 revealed a low penetration, reaching maximum at 5 h (2.5870.23 mmol g
1 fresh seeds at 120 mM) and declining thereafter to the level of water control by 8 h. This revealed the activation of antioxidants and H2O2 scavenging in seed after 5 h. Seeds treated with 1–120 mM H2O2 for 8 h and germinated in saline (150 mM NaCl) medium curtailed the mean germination time (MGT) being even less than water controls. Level of H2O2 in seedlings arising from H2O2-treated seeds grown under salinity was markedly lower than salinized controls, suggesting the operation of antioxidant system in them. These seedlings exhibited better photosynthetic capacity, particularly the stomatal conductance (gs), thus improving the leaf gas exchange due to stomatal component of photosynthesis. Moreover, H2O2 treatment improved leaf water relations and maintained turgor. Although Na+ and Cl
content increased due to salinity, H2O2-treated seedlings + + 3
displayed greater tissue K+, Ca2+, NO
3 PO4 levels and improved K :Na ratio. H2O2 treatment enhanced the membrane properties, as revealed from greatly reduced relative membrane permeability (RMP) and less altered ion leakage pattern (comparable to water controls). Seedlings exhibited the expression of two heatstable (stress) proteins with apparent molecular masses of 32 and 52 kDa. Results suggest that H2O2 signals the activation of antioxidants in seed, which persists in the

Abbreviations: Ci, substomatal CO2 concentration; E, transpiration rate; EC, electrical conductivity; gs, stomatal conductance; MGT, mean germination time; PAR, photosynthetically active radiation; Pn, net photosynthetic rate; ROS, reactive oxygen species; RMP, relative membrane permeability; cw, water potential; cs, osmotic potential; cp, turgor potential Corresponding author. E-mail address: [email protected] (A. Wahid). 0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.01.005

ARTICLE IN PRESS 2

A. Wahid et al. seedlings to offset the ion-induced oxidative damage. These changes led to the expression of stress proteins and improved physiological attributes, which supported the seedling growth under salinity. & 2006 Elsevier GmbH. All rights reserved.

Introduction Salinity has long been identified as one of the most pervasive environmental hazards, limiting crop production mostly in arid regions of the world (Pessarakli and Szabolics, 1999; Pitman and La+ uchli, 2002). Excess of ions in root medium exerts effects like osmotic strain, ion specificity/toxicity, nutritional imbalances (Wyn Jones and Gorham, 2002), changes in cell metabolites levels (Rhodes et al., 2002; Wahid and Ghazanfar, 2006) and diminished growth and yield (Ashraf and Harris, 2004; Wahid et al., 2004; Ahmad et al., 2005). Both photochemical and biochemical aspects of photosynthesis are affected by salinity (Dubey, 2005), which limit the generation of resources and/or diversion of the available resources towards stress tolerance (Ashraf and Harris, 2004). One amongst the pronounced effects of salinity is the peroxidation of lipid and loss of membranes integrity (Sairam et al., 2002). Increased cell membrane stability has therefore been taken as screening tool against salinity stress (Farooq and Azam, 2006). Other important consequence of abiotic stresses is increase in the cellular levels of reactive oxygen species (ROS), which show toxicity to the metabolic functions after conversion to H2O2 (Sairam et al., 2002; Sairam and Tyagi, 2004). There is compelling evidence about the biological activity of ROS with emphasis on the function of H2O2 as a signal molecule in plants (Dat et al., 2000; Overmyer et al, 2003; Hung et al., 2005). Chamnogpol et al. (1998) reported that H2O2 can work as an intermediate signal upstream of both ethylene and salicylic acid during plant stress responses, or can serve as a second messenger in signal transduction pathways, leading to stress acclimation (Foyer et al., 1997). Available information suggest that H2O2 directly regulates the expression of numerous genes, some of which are involved in plant defense and the hypersensitive response (Kovtun et al., 2000), antioxidants, cell rescue/defense proteins, and signaling proteins such as kinase, phosphatase, and transcription factors (Robert and David, 2004; Hung et al., 2005). Hence, H2O2 signaling is of potential significance to any program aimed at improving crop tolerance to environmental stresses.

Salt-induced production of ROS and damage caused by them is an important area of research. Various studies exhibit inter- and intra-specific differences in the production of ROS by salinity and their scavenging by the activation of antioxidants, e.g. in rice (Vaidyanathan et al., 2003), tomato organelles (Mittova et al., 2004), forage grass species (Kim et al., 2004) and wheat (Sairam et al., 2005). This shows that oxidative stress tolerance is genetically controlled and provides a wide scope for crop improvement using conventional breeding and selection, transgens production (Hung et al., 2005; Zhao et al., 2006) or adopting physiological approaches (Agarwal et al., 2005; Azevedo Neto et al., 2005). Among various strategies, pre-sowing treatment and priming of seeds are easy, low cost low risk and effective approaches to overcome the environmental stress problems (Wahid and Shabbir, 2005; Ashraf and Foolad, 2005). Priming is a controlled hydration process followed by redrying that allows metabolic activities to proceed before radical protrusion (Khan, 1992; Sivritepe et al., 2003, 2005). Various priming strategies include osmopriming, halopriming, hormonal priming or hydroprining, etc. which involving treatment of seeds with osmotica, inorganic salts, hormones or water, respectively, to induce pre-germination changes. These changes usually have profound effects on germination rate and uniformity in emergence of seedlings, specifically under stressful conditions (Parera and Cantliffe, 1991; Ashraf and Foolad, 2005). However, reports on the use of stress signaling agents like H2O2 are scarce. Exogenous application of H2O2 increases chilling tolerance by enhancing the glutathione level of mung bean seedlings (Murphy et al., 2002). Azevedo Neto et al. (2005) reported that addition of H2O2 to the nutrient solution induces salt tolerance by enhanced activities of antioxidants and reduced peroxidation of membrane lipids in leaves and roots of maize as an acclimation response. The interaction of signals conferring stress tolerance in accomplishing better crop growth and yield is a priority area of research. Better understanding of mechanism(s) that enables plant to adapt to salt stress is necessary to make the best use of saline soils. Role and mechanism(s)

ARTICLE IN PRESS Improved wheat salt tolerance by seed pretreatment with H2O2 of H2O2 as seed pretreatment in inducing salt tolerance are still elusive. Here we report some physiological and biochemical changes induced by H2O2 during seed treatment and their involvement in conferring salt tolerance upon wheat seedlings.

Materials and methods Time course changes in seed H2O2 level following soaking Healthy looking wheat (Triticum aestivum L. cv. MH-97) seeds were surface sterilized with 0.1% HgCl2 for 3 min followed by repeated washings with sterilized distilled water. Seeds (250 per replicate) were transferred to 0, 1, 40, 80 and 120 mM solution (200 mL of H2O2, contained in screw-capped bottles. After sampling from each treatment at hourly intervals for 8 h the seeds were washed with distilled water, blot dried, immediately frozen and transferred to
70 1C. Later the seed samples were determined for the absorbed H2O2 as described below. The experiment was repeated thrice.

Treatments and plant growth conditions Seeds treated with 0 (water), 1, 40, 80, and 120 mM concentration of H2O2 for 8 h, as described above, were sown in pots (30 cm diameter and 10 cm deep) containing acid and water washed sand. Experimental treatments were: no H2O2 and no salt (water control); no H2O2 and 150 mM NaCl (salinized control); H2O2-treated seeds with 1, 40, 80 and 120 mM level alone (H2O2 controls) or in combination with 150 mM NaCl (H2O2 salinized). Immediately after soaking 25 seeds were sown in each pot. Pots were kept in an illuminated growth chamber (Eyelatron, FLI-301N, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) set at 25/22 1C temperature (day/night), 475 mmol m
2 s
1 light intensity at leaf surface, 12 h day length and 50–55% relative humidity (day/night).

Germination, growth, photosynthesis and water relations Number of seedlings emerged was recorded daily. Mean time taken to germination (emergence of plumule) was computed following the formula of Ellis and Roberts (1981) when the germination was complete in water control pots. After completion of germination in all pots, uniform seedlings (thinned to 9 per pot) were grown for 15 days prior

3

to carryout determination. Leaf area of the intact plants was determined as maximum leaf length  maximum leaf width  0.70 (correction factor calibrated for all leaves). Net photosynthetic (Pn), and transpiration rates (E), stomatal conductance (gs) and substomatal CO2 concentrations (Ci) were determined of second fully expanded leaf using the infrared gas analyzer (IRGA; Analytical Development Company, Hoddesdon, England). The set of conditions for these determinations was: molar air flow per unit leaf 335 mM m
1 s
1, atmospheric pressure 99.6 kPa, PAR on leaf surface 475 mmol m
2 s
1, CO2 concentration 357 mmol mol
1 and ambient temperature 25 1C. Water potential (cw) of second fully expanded leaf was measured using pressure chamber (Pressure Chamber Arimad 2, Germany). To determine osmotic potential (cs), the freshly excised leaves from same position were quickly frozen at the time of harvesting and stored at
70 1C for a week; thawed, sap extracted and collected in a microfuge tubes, centrifuged and determined for cs using the vapor pressure osmometer (Model Wescor 5520, Utah). The turgor potential (cp) was determined from the difference of cw and cs. Fresh weight of seedlings was recorded immediately after harvesting while dry weight was taken after drying the plants in an oven at 70 1C for a week.

Relative membrane permeability and ion leakage pattern Relative membrane permeability (RMP) and pattern of ions leakage from the shoots were determined following the methods of Yang et al. (1996) and Wahid and Shabbir (2005), respectively. Excised fresh leaves from entire shoots (0.5 g) were immediately put in small Erlenmeyer flasks containing known amounts of distilled water and briefly vortexed to determine EC0. The test tubes containing leaves in distilled water were kept at 4 1C for 24 h and EC1 was determined. Half of the leachate along with tissue was taken, and the amounts of
2+ Na+, K+, Cl
, NO
were determined 3 , PO4 and Ca from leachate. The other half was autoclaved, leachate filtered and determined for EC2. RMP was computed with the formula: RMP (%) ¼ [(EC1
EC0)/(EC2
EC0)]  100.

Protein electrophoresis The frozen leaf tissue was ground in phosphatebuffered saline (pH 7.4) in the presence of acidwashed autoclaved sand and cocktail protease inhibitors, and centrifuged (Sambrook and Russell,

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A. Wahid et al.

Chemical analysis

3.0 2.5 H2O2 (µmol g-1 fresh seed)

2001). Total amount of proteins in the supernatant was determined by Bradford assay (Bradford, 1976). Heat-stable proteins were obtained by heating an aliquot from the above at 95 1C for 5 min, centrifuged and determined from the supernatant. Proteins (10 mg per lane) were separated by SDS-PAGE using 13% (w/v) acrylamide gels and stained using Coomassie brilliant blue dye. Protein markers of known molecular weight (Bio-Rad, USA) were run to ascertain the molecular weights of unknown protein bands on the gel.

2.0 1.5 1.0 0 µM e 1 µM d 40 µM c 80 µM b 120 µM a

0.5 0.0




NO
3

For the determination of Cl and contents, the dried ground whole shoots were extracted in boiling deionized water, while for Na+, K+, PO3
4 and Ca2+ the tissue was digested in a mixture of nitric and perchloric acids (3:1 ratio). The analysis from the extracted material or leachate for Na+, K+, Ca2+ was carried out by using a flame photometer and Cl
by a chloride analyzer. NO
3 was determined by the colorimetric method of Kowalenko and Lowe (1973) using chromotropic acid, and that of PO3
4 using Barten’s reagent (Yoshida et al., 1976). Hydrogen peroxide levels in seed or whole shoots were determined as described by Velikova et al. (2000).

1

2

3 4 5 6 7 Soaking time (h)

8

Figure 1. Absorption of H2O2 in seed following soaking in 0, 1, 40, 80 and 120 mM levels of H2O2. Vertical bars are standard error of means. Letters on the legends indicate significant ðPo0:05Þ difference among the H2O2 levels used to soak the seeds.

the concentrations used as revealed from the significant ðPo0:001Þ difference among the H2O2 levels of seed. At 6 h, there was a sharp decline in the absorbed H2O2 at all concentrations, which approached water control seeds within 8 h.

Germination and growth characteristics Statistical analysis In the absence of any difference ðP40:05Þ in the seedlings characteristics raised from water controls and those of H2O2 non-salinized ones, the latter were excluded from the analysis of data. Therefore, treatments included for comparisons were water controls, salinized controls and H2O2 salinized. The experiment, performed three times, was laid out in complete randomization replicated thrice. Data recorded each time were pooled for statistical analysis to determine the significance of variance while Duncan’s multiple range test was applied to find meaningful differences among treatments. Linear correlation was established between two variables where mentioned.

Results Changes in seed H2O2 following soaking Soaking in increased H2O2 levels revealed its accumulation in seeds up to first 5 h, reaching a maximum of 2.5870.23 mmol g
1 fresh seeds (Fig. 1). The accumulation was proportional to

Mean time taken to seed germination (MGT) varied considerably among the treatments ðPo0:01Þ. H2O2 applied at 40, 80 and 120 mM combined with 150 mM NaCl curtailed the MGT substantially, which was even shorter than water controls, while it was considerably delayed in salinized controls (Fig. 2). Seed treatment with H2O2 enhanced almost all the growth parameters of wheat seedlings. Shoot fresh and dry weights showed almost similar patterns of variation, being the lowest in salinized control seedlings followed by those receiving H2O2 at 1 and 40 mM levels and salinized. The highest shoot fresh and dry weight was recorded from the seedlings receiving 80 and 120 mM H2O2 followed by water control. Likewise, leaf area differed significantly ðPo0:01Þ among the treatments. It was greatest in water control seedlings and those receiving 80 mM H2O2 as seed treatment; other H2O2 treatments were less effective (Fig. 2).

Photosynthetic attributes Net photosynthetic rate (Pn) was the highest in the water control leaves which was followed by the

ARTICLE IN PRESS Improved wheat salt tolerance by seed pretreatment with H2O2

Nutrient relations Data revealed that Na+ content was the lowest in water control seedlings. Salinized control and salinized H2O2-treated shoots indicated no significant ðP40:05Þ differences in Na+ content; nonetheless, it was the highest in the former (Fig. 5). K+ content on the other hand was the highest in water

Mean germination time (days)

c

c

c

3.0 2.0 1.0

Shoot fresh weight (mg plant-1)

375.0 a

a

ab

b

300.0 b 225.0 c

150.0 75.0 0.0 45.0

a

ab 36.0

ab

27.0

a

ab

c

18.0 9.0 0.0 40.0 a

a

32.0

b

b c

24.0 d 16.0 8.0

12 0

80

40

0.0 1

Applied salinity substantially reduced leaf cw ðPo0:01Þ, but this reduction was the lowest in salinized control seedlings alone. Seed treatment with H2O2 improved this attribute considerably at 40, 80 and 120 mM H2O2 levels (Fig. 4). Leaf cs did not differ much ðP40:05Þ among the salinized control or salinized H2O2-treated seedlings, albeit it was significantly higher in water-treated control seedlings (Fig. 4). Leaf cp, highly crippled in salinized control, was greatly improved with in salinized H2O2-treated seedlings ðPo0:001Þ, which was even greater than water control ones. In this regard 80 mM H2O2 level was most effective (Fig. 4).

b

b

0

Leaf water relations

4.0

Co nt ro l

Data revealed that applied salinity increased the endogenous H2O2 content of shoots. H2O2 treatment caused a substantial reduction in endogenous H2O2 content under salinity, which was even lower than the water control shoots. H2O2-treated salinized seedlings although did not differ much ðP40:05Þ for H2O2 production, its content was low at 40 and 80 mM H2O2 levels (Fig. 3).

a

5.0

0.0

Shoot dry weight (mg plant-1)

H2O2 content of seedlings

6.0

Leaf area (cm2 plant-1)

salinized control leaves arising from seeds treated with 80 and 120 mM H2O2, while this value was the lowest in salinized controls, thus forming the basis of significant differences among the treatments ðPo0:01Þ. On the other hand, E did not differ much among the treatment ðP40:05Þ; nonetheless, the value of this parameter was highest in 80 and 40 mM H2O2-treated salinized seedlings followed by water controls (Table 1). Applied salinity substantially reduced gs, whilst the H2O2 treatment was effective in improving it up to the level of controls, thus revealing the significant ðPo0:001Þ differences among the treatments. Although there was little difference among the treatments ðPo0:05Þ Ci, it was highest in 40 mM H2O2 and salinized control seedlings. The lowest Ci was noted in the leaves of water control seedlings followed those salinized and receiving 80 mM H2O2 as seed treatments (Table 1).

5

Treatments

Figure 2. Changes in mean germination time and growth characteristics of the whole shoots arising from seeds treated with increasing H2O2 levels. In this and subsequent figures: (a) vertical bars are standard errors of means, (b) data bars denoted by same alphabets differ non-significantly ðP40:05Þ and (c) 0, 1, 40, 80 and 120 are the H2O2 levels used to soak seeds for 8 h, which were germinated in saline medium (150 mM NaCl). Controls represent the seedling arising from water-treated seeds (water controls).

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A. Wahid et al.

Table 1. Changes gas exchange and substomatal CO2 concentration of wheat leaves under increased H2O2 concentrations combined with or without salinity H2O2 levels (mM)

Salinity treatments (mM)

Net photosynthetic rate (mmol m
2 s
1)

Transpiration rate (mmol m
2 s
1)

Stomatal conductance (mol m
2 s
1)

Sub-stomatal CO2 conc (mmol mol
1)

Water control Salinized control 1 40 80 120

— 150 150 150 150 150

10.1470.68a 3.4570.12d 3.9570.41d 5.3970.45c 7.5070.86b 6.7370.67b

2.3370.18ab 2.0470.11c 2.1270.06b 2.4170.21a 2.4570.08a 2.2670.16ab

0.2470.031a 0.1570.020c 0.1970.021b 0.2170.023ab 0.2470.021a 0.2470.024a

233.3722.1b 261.0725.2a 258.3710.5a 262.3711.9a 244.7715.6b 258.3715.3a

15.0

1.0 b

c

6.0

c

c

c

3.0 0.0

a b

b

b

a

a

a

a

a

c

0.6 0.4

12 0

0.0

Treatments

1.5

Figure 3. H2O2 concentration in the whole shoots arising from seeds treated with increasing H2O2 levels. See Fig. 2 for details. ψs (-MPa)

a

b 0.9 0.6 0.3 0.0 0.6 0.5

ψp (-MPa)

a

a

0.4

b

0.3

b

0.2 0.1

0

80

12

nt

ro

l

0.0 Co

controls but lowest in salinized controls. Salinized H2O2-treated seedling, irrespective of H2O2 concentration, exhibited improved tissue K+ content. The changes in Na+ and K+ contents led to significant ðPo0:001Þ difference among the treatments revealing the effectiveness of H2O2 in enhancing the K+:Na+ ratio, which was mainly related to increased K+ content. Trend of shoot Cl
accumulation was similar to that of Na+, which again substantiated the effectiveness of H2O2 in reducing the tissue concentration of both the toxic ions (Fig. 5). Salinized control seedlings indicated the lowest 3
contents of Ca2+, NO
3 and PO4 , whilst the application of H2O2 as seed treatment was pretty effective in enhancing the content of these ions (Fig. 5). Among those, Ca2+ content, most affected in salinized controls, showed no distinct improvisation in salinized H2O2-treated seedlings. Salinized control and 1 mM H2O2 treatments although exhibited reduced content of NO
3 , H2O2 applied at higher levels, particularly at 80 mM level, was the most effective in enhancing the content of this ion, which was nearly at par with the water control

a

1.2

40

80

40

1

0.2 0

Co nt ro l

a

0.8

1

9.0

a

0

12.0

ψw (-MPa)

H2O2 (µmol g-1 FW)

See ‘‘Materials and methods’’ for details about the treatments. Means7standard error. Means denoted by same alphabet differ non-significantly ðP40:05Þ.

Treatments

Figure 4. Water relations characteristics of leaves of the plants arising from seeds treated with increasing H2O2 levels. See Fig. 2 for details.

seedlings. Changes in PO3
content of seedlings 4 were similar to NO
3 or even greater under H2O2treated seedlings under salinity (Fig. 5).

ARTICLE IN PRESS Improved wheat salt tolerance by seed pretreatment with H2O2

Heat-stable (stress) proteins Wheat seedlings raised from H2O2-treated seeds germinated in saline medium, indicated the expression of two heat-stable (stress) proteins with apparent molecular masses of 32 and 52 kDa. The expression of both these proteins was evident at all levels of H2O2; nonetheless, their expression was stronger at 80 mM level (Fig. 7). Such protein expression was not evident in water- or salt-treated control shoots.

Na+ (mg g-1 DW)

a

a

a

a

b

b

b

b

b

b

b

b

12.0

a

b

9.0 6.0 3.0 0.0

a

K+ (mg g-1 DW)

40.0

c

30.0 20.0 10.0 0.0 8.0

a

K+:Na+ ratio

6.0 4.0 c 2.0 0.0

Cl- (mg g-1 DW)

30.0

a

a

24.0

b

c

b

18.0 d

12.0 6.0 0.0 15.0

Ca2+ (mg g-1 DW)

RMP, an index of stress sensitivity, was greatly increased due to salinity, whilst seed treatment of H2O2 at all levels reduced it under salt stress; 80 and 120 mM levels were the most effective (Fig. 6). While RMP was increased, the patterns of ion leakage also varied considerably. Na+ and Cl
being prevalent ions in the medium were leaked in major quantities, but their leakage in the control shoots was minimal. Surprisingly, the potassium leakage in the water control seedlings matched the salinized ones, while H2O2 pretreatment substantially reduced its leakage. K+ leakage from H2O2-treated seedlings was the lowest among all the ions. 3
Leakage of Ca2+, NO
followed almost 3 and PO4 similar pattern, notwithstanding the differences in their content in the leachates (Fig. 6). Ca2+ leakage was lowest in water control shoots, but various H2O2 treatments did not indicate much difference under salinity. NO
3 leakage was the most pronounced in the salinized control seedlings, while it was significantly ðPo0:01Þ less in H2O2-treated salinized ones and lowest in water controls. Likewise, PO3
leakage was lowest in water controls, 4 followed by 1, 80, 40 and 120 mM H2O2 levels, but highest in salinized controls (Fig. 6).

15.0

a

12.0 9.0

b

c

c

b

c

6.0 3.0 0.0 35.0

NO3- (mg g-1 DW)

Relative membrane permeability and ion leakage pattern

7

a b

28.0 c

21.0

ab

b

c

14.0 7.0 0.0

a

ab

12 0

ab

80

b

10.0 5.0

40

1

0

ro

l

0.0 nt

Exogenous use of various chemicals to reduce the adverse effects of stresses has great implications both from theoretical and practical perspectives (Uchida et al., 2002; Sivritepe et al., 2003, 2005). H2O2 is a strong oxidizing agent that injures cells and damages photosynthesis at high concentration when produced internally or applied externally (Foyer et al., 1994; Samuilov et al., 2001; Sairam et al., 2002), but acts as stress signal in low concentrations (Foyer et al., 1997; Dat et al., 2000; Desikan et al., 2004). Soaking of seeds in

ab

15.0

Co

Discussion

PO43- (mg g-1 DW)

20.0

Treatments

Figure 5. Ionic content of whole shoots arising from seeds treated with increasing H2O2 levels. See Fig. 2 for details.

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A. Wahid et al. 25.0

a

RMP (%)

20.0

b

b

15.0

b c

bc

b

b

b

d

cd

b

cd

d

c

b

abc

ab

10.0 5.0 0.0

Na+ (mg L-1)

20.0

a b

15.0 c

10.0 5.0 0.0

K + (mg L-1)

30.0

a

a

c

24.0 18.0 12.0 6.0 0.0

Cl- (mg L-1)

35.0

a

28.0 21.0

c

14.0 7.0 0.0

Ca2+ (mg L-1)

35.0

a

28.0 d

21.0

abc

bcd

14.0 7.0 0.0

27.0

b

b

b

b

40

a

36.0

1

NO3- (mg L-1)

45.0

b

b

c

18.0 9.0 0.0 35.0

PO43- (mg L-1)

a 28.0

b

b

c

21.0 14.0 7.0

12 0

80

0

Co

nt

ro l

0.0

Treatments

Figure 6. Relative membrane permeability (RMP) and patterns of ion leakage from whole shoots arising from seeds treated with increasing H2O2 levels. See Fig. 2 for details.

increased H2O2 levels (1–120 mM) for 8 h and its subsequent scavenging (Fig. 1) revealed the activation of antioxidants. Seed absorption revealed a low penetration of H2O2, which supports the view that it can effectively modulate gene expression when used in lower amounts (Bailly et al., 2002; Hung et al., 2005; Azevedo Neto et al., 2005). Use of seed pretreatment is advantageous only when it supports the emergence and establishment of seedling. Initial absorption and later scavenging of H2O2 in the seed provide evidence about the occurrence of changes mainly related to protection against oxidative damage. Therefore, we evaluated the effects of H2O2 produced changes on the salinity tolerance in terms of germination, growth, physiological attributes and expression of stress proteins in salinized H2O2-treated seedlings of wheat and compared with water control and salinized control ones. Increased salinity has multifarious effects on the cell metabolism; production and damage by ROS is one amongst those (Sairam et al., 2002; Agarwal and Pandey, 2004; Mittova et al., 2004). Treatment of seed with increased H2O2 levels produced no visible symptoms of toxicity; rather it alleviated the deleterious effects of salinity on seed germination and seedling growth, as evidenced by a significantly curtailed MGT or minimal reduction in most growth parameters of salt-treated seedlings (Fig. 2). Likewise, effects of salinity on both light and dark reactions of photosynthesis are well established (Dubey, 2005). Among gas exchange characteristics Pn and gs are more sensitive to salt stress, while photosynthetic protection against salt-induced ROS by increased activity of antioxidants has been suggested in cotton (Meloni et al., 2003) and tomato (Herbette et al., 2005). Despite this, studies to show the improvement in photosynthetic capacity under stress conditions as a result of treatment with H2O2 are scanty. It is noteworthy that improved gs and sustained Pn and E in H2O2-treated seedlings under salinity were of great importance in growth and dry matter production (Table 1). These parameters when paralleled with shoot dry weight revealed that improved gs (r ¼ 0.962**; n ¼ 6) was the most important of all photosynthetic attributes. Sustained leaf gas exchange capacity under salinity is crucial and H2O2 produced changes maintained gs that enabled the leaves to display high Pn and shoot dry mass. Comparison of water control and H2O2-treated salinized seedlings revealed substantially lowered shoot H2O2 content of the latter (Fig. 3). This further substantiated the effectiveness of activated antioxidants to scavenge the H2O2. These changes curtialed the time taken to germination, improved gas exchange capacity

ARTICLE IN PRESS Improved wheat salt tolerance by seed pretreatment with H2O2

9

Figure 7. Expression of heat-stable (stress) proteins in whole shoots treated with increasing H2O2 levels. Arrows indicate the expression of a 32 kDa (lower) and 52 kDa (upper) bands. See Fig. 2 for details.

and vigorous growth of seedlings under salinity. This conforms to the notion that ROS-activated accumulation of stress related genes transcripts is spread over 2 days of H2O2 application (Uchida et al., 2002). Excess ion-induced water deficit is among the other effects of salinity, which is more deleterious to the mobilization of reserves, radical protrusion and early growth of seedlings (Dood and Donovan, 1999; Wahid et al., 1999). Direct H2O2 application to growing plants is lethal due to its strong oxidizing property (Foyer et al., 1994). Nonetheless, it improves the leaf water balance as indicated by ABA content as well as induction of antioxidant system (Xing et al., 2004; Kukreja et al., 2005). In line with these findings, as a result of activation of antioxidants, H2O2-treatment improved the water relations of salinity-treated seedlings by turgor maintenance, which was comparable to the water control seedlings (Fig. 4). In addition, excess of toxic ions cripples the nutritional status of plants by ion specificity or induced nutrient deficiency (La ¨uchli, 1986; Wyn Jones and Gorham, 2002). Seed treatment with inorganic and organic agents greatly reduces the stress effects and enhances the essential nutrient content (Sivritepe et al., 2003, 2005; Wahid and Shabbir, 2005). This study revealed the improved nutrient status of shoots raised from H2O2-treated seeds, albeit there was a build up in the level of both Na+ and Cl
in

the shoots due to high substrate salinity. H2O2 pretreatment of seed, particularly at higher levels, helped reduce the build up of Na+ and Cl
and 2+ 3
improved K+, NO
content, and K+:Na+ 3 , PO4 , Ca ratio in most cases (Fig. 5). Increased tissue K+, 2+ 3
NO
contents and K+:Na+ ratio are 3 , PO4 , Ca important to metabolic activities, and are therefore taken as valid physiological criteria of salt tolerance (Ashraf and Harris, 2004). These findings explicitly show that H2O2 triggered changes, primarily linked to the activation of antioxidants, stood fast in maintaining turgor and meeting plant nutritional requirment to thrive under salinity. Stability of biological membranes has been taken as a screening tool to assess the salinity stress effects (Kukreja et al., 2005; Farooq and Azam, 2006). Salinity has a pronounced effect on the peroxidation of membrane lipids, enhances their permeability and modulates the patterns of ions leakage (Sairam et al., 2002; Kukreja et al., 2005). Seed treatment with H2O2, in this study, reduced the RMP and leakage of ions like, K+, Ca2+, NO
3 and PO34, which was not much different from the water control seedlings in most cases (Fig. 6). Production of ROS, including H2O2, is a noxious factor in the peroxidation of membrane lipids (Sairam et al., 2002; Sairam and Tyagi, 2004). A low H2O2 content of seedlings arising from H2O2-treated seeds (Fig. 3), as result of reduced oxidative damage, is a plausible justification for improved membrane

ARTICLE IN PRESS 10 integrity and consistently low leakage of important ions. Expression of stress proteins is an important adaptive strategy of environmental stress tolerance. They are highly water soluble and heat stable, associate to cytoplasmic membranes and organelles and act as molecular chaperones, etc. (Schoffl et al., 1999; Sanmiya et al., 2004; Wahid and Close, 2006). Seedlings arising from H2O2treated seeds indicated the expression of two heat-stable (stress) proteins with apparent molecular masses of 32 and 52 kDa (Fig. 7). Expression of these proteins was strongest at 80 mM H2O2; the most effective level in mitigating salinity effect for most attributes of wheat seedlings. Earlier studies demonstrated the expression of small heat shock proteins (HSPs), including mitochondrial HSP22, as an acclimation response to H2O2 application in tomato (Banzet et al., 1998) and Arabidopsis (Pnueli et al., 2003). In view of their properties of associating to the cellular membrane and organelles, specific expression of stress proteins is an important adaptive manifestation in maintaining the integrity, native configuration and topology of membranes components to ensure their normal functioning under salinity stress. In conclusion, H2O2 signaling in the activation of antioxidants in seed and subsequently seedlings has beneficial effects on the growth and physiological phenomena by prevention of oxidative damage and enhanced capacity of wheat to withstand high salinity. Maintenance of membrane integrity and expression of stress proteins are important manifestations of seed pretreatment with H2O2 under salinity. Such responses may be of considerable value in the development of improved method for crop protection against environmental stresses.

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