Physiological Responses of Krishum (Iris Lactea Pall. Var. Chinensis Koidz) to Neutral and Alkaline Salts Y

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J. Agronomy & Crop Science (2008) ISSN 0931-2250

SALINITY STRESS Physiological Responses of Krishum (Iris lactea Pall. var. chinensis Koidz) to Neutral and Alkaline Salts Y. Wang1,2, J. X. Guo1, Q. L. Meng1 & X. Y. Cui1

1 Grassland Research Institute, Key Laboratory of Vegetation Ecology, Northeast Normal University, Changchun, China 2 College of Urban and Environmental Science, Northeast Normal University, Changchun, China

Keywords Abstract alkaline salt stress; antioxidase activities; biomass; ion contents; krishum (Iris lactea Pall. The aims of this study were to compare the physiological responses of krishum var. chinensis Koidz); neutral salt stress (Iris lactea Pall. var. chinensis Koidz) to neutral and alkaline salt stress and identify and examine the mechanisms involved in plant response to salt treat- Correspondence ments. In this study, biomass, ion accumulation (Na+,K+,Ca2+,Mg2+), Jixun Guo organic solute (proline) concentration, rate of membrane electrolyte leakage Grassland Research Institute (REL) and antioxidase activities including those of superoxide dismutase Key Laboratory of Vegetation Ecology Northeast Normal University (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and peroxidase (POD, EC 5268 Renmin Street, Changchun 1.11.1.7) were investigated in krishum under different concentrations of NaCl, China Na2CO3 and the mixture of the two salts in the same volume. All three treat- Tel.: +86-0431-85098937 ments caused increases in Na+ concentration, proline content and REL and Fax: +86-0431-85095065 decreases in root Mg2+ and K+ content. Increased Ca2+ and antioxidase activi- E-mail: [email protected] ties were observed at lower external Na+ concentrations. However, at higher external Na+ levels, decreased Ca2+ and antioxidase activities were detected. Accepted July 30, 2008 Alkaline salt resulted in more damage to krishum than neutral salt including doi:10.1111/j.1439-037X.2008.00337.x lower SOD, POD and CAT activities and decreased proline content, relative to neutral salt. High Na+ and low K+ in krishum intensiﬁed ion toxicity under alkaline condition. Alkaline salt caused greater harm to plants than neutral salt, the primary reason of which might be the lower Ca2+ content in the plant under alkaline salt stress.

widely distributed in northern China, Tibet, eastern Introduction Russia and Mongolia (Xu et al. 2002). Medicinal value, High salinity soils have major ecological effects on plant propagation, ornamental potential and species morphol- growth and productivity (Giridara Kumar et al. 2003). ogy have been investigated in several studies (Fu et al. Saline soils comprise 831 million hectares on earth and 2001, Wang 2002, Yang 2002, Mou et al. 2006). Huang saline–alkaline soils occupy 434 million hectares (Jin et al. et al. (2005) discussed the effects of neutral salt stress on 2006). The main characteristics of these soil types include growth and osmotic adjustments in krishum. Bai and Li + ) 2) high levels of Na and Cl , and CO3 is often a (2005) further examined the impacts of salt stress on co-occurring anion. growth and K+ and Na+ absorption/transportation. Plants poorly adapted to these ecosystems may suffer However, little research has addressed the species’ not only sodium and chlorine toxicity but also pH stress tolerance to alkaline salts. (Jin et al. 2006). Plants under high salinity regimes dem- High salt concentrations result in cellular ion imbal- onstrate speciﬁc adaptations for survival and reproductive ance, which can lead to ion toxicity, osmotic stress and success. Krishum (Iris lactea Pall. var. chinensis Koidz), a production of reactive oxygen species (ROS) (Ashraf perennial monocotyledon and a member of the Iridaceae, 1994, Mittler 2002). As a primary stress factor, high NaCl is a plant well adapted to saline–alkaline soil conditions. uptake competes with the uptake of other nutrient ions, Typically, the species occupies patchy distributions and is especially K+, leading to K+ deﬁciencies (Parid and Das

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2005). Increased NaCl treatments induce increases in Na+ Hoagland nutrient solution (Arnon and Hoagland 1940). and decreases in Ca2+,K+ and Mg2+ levels in a number The experiment was carried out in a naturally lit glasshouse of plant species (Khan et al. 1999, 2000, Khan 2001). at the Department of Life Science, Northeast Normal Uni- Plants possess a number of antioxidant enzymes, versity. Eighty days following emergence, seedlings were such as superoxide dismutase (SOD; EC 1.15.1.1), transplanted into 48 plastic pots (20.5 cm diameter and catalase (CAT, EC 1.11.1.6) and peroxidase (POD; EC 20.0 cm deep) containing 5.0 kg of well-washed dry sand. 1.11.1.7). These three enzymes protect cells against the Twenty seedlings were planted in each pot. Ten days after deleterious effects of ROS. NaCl-induced antioxidant transplanting, seedlings were separated into four groups enzyme activities have been reported in cotton (Gossett and watered with the same nutrient solution as above (Ar- et al. 1994), wheat (Meneguzzo et al. 1999), rice non and Hoagland 1940). Three treatments plus a non-sali- (Dionisio-Sese and Tobita 1998), pea (Hernandez et al. nized control were compared. Three nutrient solution 1995) and wild tomato (Mittova et al. 2002). When treatments were prepared as follows: NaCl (25, 50, 100, 200

ROS is synthesized during stress responses, the induc- and 400 mm); Na2CO3 (12.5, 25, 50, 100 and 200 mm); tion of ROS-scavenging enzymes is the most common and mixture of the same volume of NaCl and Na2CO3 solu- mechanism for detoxification (Sairam and Saxena 2000; tions (Mix) (25 mm NaCl + 12.5 mm Na2CO3;50mm Mittler 2002). NaCl + 25 mm Na2CO3; 100 mm NaCl + 50 mm Na2CO3; Studies suggest that NaCl leads to high proline accu- 200 mm NaCl + 100 mm Na2CO3; and 400 mm + mulation (Khatkar and Kuhad 2000, Singh et al. 2000), NaCl + 200 mm Na2CO3). The final Na gradient levels in which can lower plant water potential. Subsequently, the three treatment groups were 25, 50, 100, 200 and plants take up more water from the environment (Girid- 400 mm (Table 1). Three replications for the control and ara Kumar et al. 2003). Although studies examining free all three treatments were conducted. proline content did not report appreciable increases under NaCl conditions (Dix and Pearce 1981, Jain et al. 1987), Determination of dry weight and preparation of proline accumulation is still considered one of several biochemical analysis plant adaptations to salinity and water deficits (Giridara Kumar et al. 2000, Ramanjulu and Sudhakar 2000, 2001). Plant samples were collected following two weeks of treat- Studies of the effects of neutral salt (NaCl) stress on ments. To determine dry weight (DW), plants were sepa- plants in saline–alkaline soils exclude information on rated into shoots and roots, and oven-dried at 60 C plant growth in the basic pH of alkaline soils and have until a constant weight was reached. Fresh shoot samples not provided comprehensive data from natural ecosys- from each plant were immediately frozen in a )75 C

tems. The stress imposed by Na2CO3 toxicity was greater ultra-cold freezer until biochemical analysis was per- than that of NaCl in Puccinellia tenuiflora Scribn. (Shi formed (see below). and Zhao 1997) and Helianthus annuus L. (Shi and Sheng 2005). However, the mechanisms of salt stress toxicity Determination of plant tissue mineral elements remain elusive. In this study, krishum biomass, ion accumulation Dried plant samples were powder-homogenized and 0.5 g (Na+,K+,Ca2+,Mg2+), proline accumulation, rate of was subsequently boiled in 10 ml of distilled water at membrane electrolyte leakage (REL) and antioxidant 100 C for 2 h using a dry heat bath. The hot water enzymes including SOD, CAT and POD imposed by extract was cooled and filtered using Whatman No. 42 fil-

NaCl, Na2CO3 and a mixture of the two salts were inves- ter paper (Khan et al. 2000). Plant tissue extracts were tigated. The aims of this study were to compare the phys- assayed for Na+,K+,Ca2+ and Mg2+ by atomic absorp- iological responses of krishum to neutral and alkaline salt tion spectrophotometry (220FS, Varian Australia Pty Ltd, stress and identify and examine the mechanisms involved Mulgrave, Victoria, Australia). in plant response to salt treatments.

Table 1 pH of the three treatment groups Material and Methods Na+ concentration Plant materials and experimental treatments 25 mM 50 mM 100 mM 200 mM 400 mM Krishum seeds were collected from Changlin County in Jinlin Province, China. Seeds were sown in plastic contain- NaCl 7.08 7.11 7.20 7.29 7.34 Na CO 11.14 11.25 11.33 11.41 11.46 ers (20.5 cm diameter and 20.0 cm deep) with washed 2 3 Mix 10.77 10.91 10.97 11.07 11.10 sand. The seeds were watered on alternate days with

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The CAT reaction solution (3 ml) contained 50 mm Proline content determination phosphate buffer (pH 7.0), 5.9 mm H2O2, 0.1 ml of Fifty milligrams of dried plant samples was homogenized enzyme extract and the reaction initiated by enzyme in 3 % aqueous sulphosalicylic acid at 100 C for 15 min. extract addition. At 240 nm, the changes in reaction solu- The homogenates were subsequently centrifuged at tion absorbance were read every 20 s. One unit of CAT 10 000 g, and proline content determined following the activity was deﬁned as an absorbance change of 0.01 unit ) procedure of Bates et al. (1973). The concentration was min 1. POD reaction solution (3 ml) contained 50 mm

expressed as milligrams perÆgram dry weight. phosphate buffer (pH 5.0), 20 mm guaiacol, 40 mm H2O2 and 0.1 ml of enzyme extract. At 470 nm, changes in reaction absorbance solution were determined every 20 s. Membrane permeability determination One unit of POD activity was deﬁned as an absorbance ) Electrolyte leakage (REL) was determined using the change of 0.01 unit min 1. Enzyme activity was expressed method of Lutts et al. (1996). One gram of fresh root and on a protein basis and crude protein extract concentra- leaf segments (1 cm in length) was collected from each pot tion was measured following the method of Bradford and washed three times with deionized water to remove (1976). For the purposes of this study, NaCl was deﬁned

surface-adhered electrolytes. Plant segments were divided as a neutral salt and Na2CO3 was defined as an alkaline equally and placed in two closed vials containing 20 ml of salt because of its higher pH in solution. deionized water. One vial was incubated at 25 Cona rotary shaker for 3 h. The electrical conductivity of the Statistical analysis solution (EC1) was determined with a conductivity gauge (DDS-307, Shanghai Precision & Scientific Instrument Co. Data were compared using the spss 11.5 statistical pro- Ltd., Shanghai, China). The second vial was autoclaved at gram (SPSS Inc., Chicago, IL, USA). Duncan post hoc 120 C for 20 min and the electrical conductivity of the tests were performed when significant differences

solution (EC2) was determined following a 25 C equili- occurred at the 5 % level. Microsoft Excel 2003 was bration. REL was deﬁned as follows (Shi and Sheng 2005): employed to generate ﬁgures.

EC REL ¼ 1 100 EC2 Results Enzyme assays Biomass and proline content Antioxidant enzyme extraction Dry biomass (DW) gradually decreased with increasing m + Fresh leaves (0.5 g) were ground in 3 ml of 50 m cold external Na under NaCl, Na2CO3 and Mix treatments phosphate buffer (pH 7.8) and centrifuged at 15 000 g (Fig. 1). External Na+ had no significant effect on DW at for 20 min at 4 C. The supernatant was used to deter- 25 or 50 mm. However, a significant reduction in DW mine enzyme activity (Raza Sayed et al. 2007). was observed at external Na+ concentrations from 100 to 400 mm, particularly at 400 mm (P < 0.01). In addition, Superoxide dismutase, catalase and peroxidase determination significant differences among treatments were indicated + The inhibition of photochemical reduction of nitroblue (P < 0.01). Na2CO3 treatment at the same external Na tetrazolium (NBT) by SOD was determined following concentrations showed the highest reduction in DW. the method of Raza Sayed et al. (2007). A reaction solution (3 ml) containing 50 lm NBT, 1.3 lm ribofla- 6 vin, 13 mm methionine, 75 mm ethylenediaminetetraace- m ) 5 tic acid (EDTA), 50 m phosphate buffer (pH 7.8) and –1 50 ll of enzyme extract was prepared and placed in 4 test tubes under 15-W fluorescent lamps for 15 min. At 3 560 nm, the irradiated solution absorbance was determined using a spectrophotometer (754 UV/VIS Spectro- 2 1

photometer, Shanghai Precision & Scientiﬁc Instrument Dry biomass (g pot Co. Ltd.). One unit of SOD activity was deﬁned as the 0 amount of enzyme that caused a 50 % photochemical 0 25 50 100 200 400 Na+ (mM) reduction inhibition of NBT. Catalase (CAT) and peroxidase (POD) activity was Fig. 1 Dry biomass of krishum under different Na+ levels ( NaCl;

measured using the method of Raza Sayed et al. (2007). Na2CO3; Mix).

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Compared with the control, DW was reduced by 23.0 %, 0.12 43.9 % and 32.4 % at 400 mm external Na+ under NaCl,

Na2CO3 and Mix treatments, respectively. The order of 0.09 + stress effect on krishum DW was Na2CO3 > Mix > NaCl. DW) –1 Proline content in both leaves and roots of krishum 0.06 + increased with increasing Na concentrations and the Leaf Na 0.03 greatest values were detected at 400 mm (Fig. 2). Among (mmol g the three treatments, leaf proline content showed signiﬁ- 0 cant differences at 50, 100, 200 and 400 mm external Na+ 0 25 50 100 200 400 (P < 0.01), while signiﬁcant differences in root proline Na+ (mM) content were observed at 100, 200 and 400 mm external 0.8 Na+ concentration (P < 0.05). Proline accumulation in leaves was 4.48, 2.24 and 2.46 times of that of the control 0.6

and 1.67, 1.27 and 1.38 times greater in roots under + DW)

m –1 NaCl, Na2CO3 and Mix at 400 m , respectively. NaCl 0.4

induced the highest proline levels, while Na2CO3 had the Leaf K least effect. 0.2 (mmol g

0 Ion contents 0 25 50 100 200 400 Na+ (mM) Na+ content in both leaves and roots increased as + external Na concentration increased (Figs 3 and 4). The 0.2 highest Na+ values were demonstrated at 400 mm. Com- pared with the control, at 400 mm external Na+ concen- 0.15 2+ tration, leaf Na+ was 2.61, 3.91 and 3.86 times higher DW) –1 0.1 under NaCl, Na2CO3 and Mix treatments, respectively. + Root Na content was increased by 1.11, 1.69 and 1.31 Leaf Ca 0.05 (mmol g

24 0 0 25 50 100 200 400 20 Na+ (mM) 16 0.16 DW)

–1 12 0.12 2+ Leaf proline

8 DW) (μg mg –1 0.08 4 Leaf Mg 0 0.04 0 25 50 100 200 400 (mmol g Na+ (mM) 0 1 0 25 50 100 200 400 Na+ (mM) 0.8 Fig. 3 Ion contents in krishum leaves under different Na+ levels

( NaCl; Na2CO3; Mix).

DW) 0.6 –1 0.4 times under the same parameters. Signiﬁcant differences Root proline

(μg mg + 0.2 in both leaf and root Na levels were found at 200 (P < 0.05) and 400 mm (P < 0.01) external Na+ concen- 0 trations. Among the three treatments, leaf Na+ content 0 25 50 100 200 400 showed signiﬁcant differences at 100 (P < 0.01), 200 Na+ (mM) (P < 0.05) and 400 mm external Na+ (P < 0.01), while + Fig. 2 Proline contents in krishum under different Na+ levels ( NaCl; signiﬁcant differences in root Na content were observed + Na2CO3; Mix). at 200 and 400 mm external Na concentration

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0.15 Compared with the control, K+ levels in leaves and roots were signiﬁcantly lower at the external Na+ higher than 0.12 50 and 100 mm, respectively (P < 0.05). The highest

+ + + DW) 0.09 decrease in K was detected at an external Na concentra- –1 tion of 400 mm. At 400 mm external Na+, leaf K+ content 0.06

Root Na was decreased by 34.4 %, 44.1 % and 39.4 % under NaCl, + (mmol g 0.03 Na2CO3 and Mix treatments and root K content was decreased by 34.1 %, 45.6 % and 39.2 %, respectively. 0 + 0 25 50 100 200 400 Among the three treatments, leaf K content showed sig- Na+ (mM) niﬁcant differences at 50 (P < 0.05), 100 (P < 0.05), 200 (P < 0.01) and 400 mm (P < 0.05) external Na+, while + 0.08 signiﬁcant differences in root K content were observed at 200 (P < 0.01) and 400 mm (P < 0.05) external Na+ + 0.06 concentration. At high external Na concentration, + DW) Na2CO3 treatment had the greatest effect. K content in –1 0.04 krishum treated with NaCl was higher than that of

Root K+ Na2CO3 and Mix (NaCl > Mix > Na2CO3). 0.02 2+ (mmol g Ca contents in leaves and roots were the highest at 50 mm external Na+ under all treatments, with the excep- 0 0 25 50 100 200 400 tion of leaf Ca2+, which was the highest for the 100 mm Na+ (mM) NaCl culture (Figs 3 and 4). Among the three treatments, leaf Ca2+ content showed signiﬁcant differences at 100, + 0.15 200 and 400 mm external Na (P < 0.05), while signiﬁ- cant differences in root Ca2+ content were observed at 50 (P < 0.05), 100, 200 and 400 mm (P < 0.01) external Na+

2+ 0.1 DW) 2+

–1 concentration. Ca content in krishum decreased at the higher external Na+ concentrations, but it was compara-

Root Ca 0.05 tively higher in NaCl, followed by Mix and lower in (mmol g Na2CO3 treatment both in leaves and roots. 2+ 0 No signiﬁcant differences in leaf Mg content among 0 25 50 100 200 400 the three treatments were observed under all the external Na+ (mM) Na+ concentrations (Figs 3 and 4). However, signiﬁcant 0.15 differences in Mg2+ levels were found in roots (P < 0.01). A reduction in root Mg2+ content with external Na+ under Na CO (r2 = 0.8996, P < 0.01) and Mix 2+ 0.1 2 3 DW) 2

–1 (r = 0.8799, P < 0.01) treatments was indicated. Root Mg2+ level was increased by NaCl treatment from 25 to 0.05 Root Mg 50 mm, decreased at 100 mm and was similar to the con- (mmol g trol at 400 mm NaCl. Among the three treatments, leaf 0 Mg2+ content showed no signiﬁcant difference at any 0 25 50 100 200 400 external Na+, while signiﬁcant differences in root Mg2+ Na+ (mM) content were observed at 25, 50, 100, 200 and 400 mm + 2+ Fig. 4 Ion contents in krishum roots under different Na+ levels (P < 0.01) external Na concentration. Mg content in

( NaCl; Na2CO3; Mix). roots under NaCl treatment was higher than that under the other two treatments at the same external Na+ level.

(P < 0.01). At the same Na+ concentrations, both leaf Enzyme activities and electrolyte leakage rate Na+ and root Na+ content increased in the order of

Na2CO3 > Mix > NaCl. Superoxide dismutase, POD and CAT activities increased In contrast to Na+ and K+ levels decreased with with external Na+ concentrations from 25 to 200 mm, increasing Na+ content in both leaves and roots (Figs 3 while 400 mm external Na+ resulted in an inhibitory and 4). External Na+ had no signiﬁcant effects on leaf K+ effect (Fig. 5). Among the three treatments, signiﬁcant content at 25 mm and root K+ content at 25 or 50 mm. differences were found at 50 (P < 0.05), 200 and 400 mm

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60 6 5 4 40 3

protein) 2 –1 SOD 20 Leaf REL (%) 1 0 (U mg 0 25 50 100 200 400 Na+ (mM) 0 0 25 50 100 200 400 60 Na+ (mM) 50 40 120 30 20 90

Root REL (%) 10 0 protein) 60 0 25 50 100 200 400 –1 POD Na+ (mM) 30 (U mg Fig. 6 Electrolyte leakage rate (REL) in krishum under different Na+

0 levels ( NaCl; Na2CO3; Mix). 0 25 50 100 200 400 Na+ (mM) Discussion 120 Biomass is a comprehensive indicator of environmental 90 stresses. Plants heavily impacted by stress demonstrate reduced biomass (Giridara Kumar et al. 2003). The results protein) 60 –1

CAT of this study indicated that a greater decrease in plant biomass was induced by alkaline salt stress than by neu- 30 (U mg tral salt stress. Therefore, alkaline salt has a higher level of 0 toxicity than neutral salt. Our ﬁndings are consistent with 0 25 50 100 200 400 results in wheat growing in calcareous soil (Nuttall et al. + Na (mM) 2003), and Puccinellia tenuiﬂora (Poaceae) (Shi and Zhao Fig. 5 Superoxide dismutase (SOD), catalase (CAT) and peroxidase 1997), onion (Sharma et al. 2001), eucalyptus (James (POD) activities in krishum leaves under different Na+ levels ( NaCl; et al. 2002) and pea (El and Shaddad 1996) under salt

Na2CO3; Mix). stress conditions. Proline is widespread in higher plants and its accu- (P < 0.01) external Na+ for SOD; at 25, 50, 100 and mulation is a characteristic of plants under saline, 400 mm (P < 0.05) for POD; and 200 (P < 0.05) and drought and freezing conditions (Ashraf 1994). Proline 400 mm (P < 0.01) for CAT. NaCl treatment induced the is osmotically active, which contributes to membrane highest SOD, POD and CAT activity, followed by Mix stability (Gadallah 1999) and mitigates the effect of + + and Na2CO3 at the same external Na level. Na on cell membrane disruption (Mansour 1998). Increases in external Na+ concentrations resulted in a Proline does not suppress enzyme activity even at rise in REL with the highest values observed at 400 mm supra-optimal levels (Wyn Jones et al. 1984). Currently, (Fig. 6). External Na+ at 25 mm did not significantly a number of studies support the hypothesis that a posi- affect leaf REL; similarly, root REL was not significantly tive correlation between proline accumulation capacity different at 25 and 50 mm. The differences in leaf REL and degree of salt tolerance operates in plants (Gzik among the three treatments were significant at 100 mm 1995). Our results support this hypothesis and indicate external Na+ (P < 0.01) and root REL displayed signifi- that plants possess greater tolerance to neutral rather cance at 200 and 400 mm external Na+ (P < 0.05). Higher than alkaline salts, consistent with a study by Shi and

REL was detected under Na2CO3 treatment with the same Sheng (2005). external Na+ concentration as the other two treatments. High Na+ concentration is known to cause osmotic

In other words, Na2CO3 imposed a greater stress injury stress, ion toxicity, nutritional imbalance or a combina- on plasma membranes than the other two conditions tion of these effects (Ashraf et al. 2001). K+ is a funda-

(Na2CO3 > Mix > NaCl). mental co-factor for many enzymes to maintain osmotic

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balance; therefore, plants require high levels of this essen- salt-stressed plants than in alkaline salt-stressed plant. tial element (Mahajan and Tuteja 2005). Consequently, Therefore, krishum had higher resistance to neutral salt higher plant K+ and lower Na+ (higher K+/Na+) levels stress than to alkaline salt stress. Furthermore, alkaline under high external Na+ conditions are beneficial to plant salt also caused higher Na+ accumulation but lowered K+ growth (Ashraf and Khanum 1997). Common proteins and Ca2+ build-up, associated with lower SOD activity. transport Na+ and K+ and Na+ competes with K+ for Although the roles of Na+ or K+ in SOD regulating activi- intracellular influx (Amtmann and Sanders 1999). In ties remained unclear, the pivotal role of Ca2+ in antioxi- addition, many K+ transport systems have some affinity dant enzyme signal transduction was evident. Agarwal for Na+. The results of this study revealed higher K/Na in et al. (2005) reported that external application of Ca2+ in krishum treated by neutral salt than alkaline salt, consis- a rooting culture caused an increase in SOD activity. tent with a study of Leymus chinensis (Poaceae) stressed Chen and Li (2001) suggested that Ca2+ acts as a second-

by NaCl and Na2CO3 (Wang et al. 1994). Thus, higher ary messenger and causes a transient increase in H2O2. + pH intensified Na transport affinities and consequently This decrease in H2O2 induces increase in antioxidant increased plant Na+ toxicity. Furthermore, our research enzyme activities leading to a long-term decrease in ROS. revealed that high pH increased cell membrane damage, Therefore, in krishum treated by alkaline salt, higher pH which also resulted in higher Na+ levels in alkaline may decrease Ca2+ accumulation and play a pivotal role stressed krishum. in reducing SOD activities. The three treatments employed in this study demon- In summary, alkaline salt was more harmful to kris- strated an increase in Ca2+ at 25 and 50 mm external Na+ hum than neutral salt. Alkaline salt caused lower SOD, concentrations. Aziz et al. (1999) also found similar POD and CAT activities than neutral salt. In addition, results in tomatoes (Aziz et al. 1999). However, leaf Ca2+ compared to neutral salt, a decrease in proline content accumulation in Glycine tabacina and G. tomentella was and selective ion absorption was observed. High Na+ not affected by increasing Na+ (Kao et al. 2006). Lynch and low K+ in krishum intensified ion toxicity under et al. (1989) suggested that a rapid increase in cytosolic alkaline conditions. Alkaline salt caused a greater harm Ca2+ under salt stress indicated a general stress signal in to plants than neutral salt, the primary reason of which plants (Lynch et al. 1989). In addition, it was determined might be the lower Ca2+ content in the plant under that Ca2+ also protects plants against the adverse effects alkaline salt stress. of Na+ and enhances growth under saline conditions (Cramer et al. 1990). In our experiments, higher Ca2+ Acknowledgements content was revealed under neutral salt conditions and not under higher pH regimes. A decrease in Ca2+ solubil- This research work was supported by grants from the ity with an increase in pH might have occurred, which National Key Basic Research Program (2007CB106801), the led to decreased absorbable Ca2+ available to krishum. In National Natural Science Foundation of China (No. addition, studies have reported that the inhibitory effects 30590382 and No. 30570273) and the Science Foundation of Na+ were reduced with increased Ca2+ concentrations. for Young Teachers of Northeast Normal University, China This was likely because of a decrease in Na+ content, (No. 20070502). which subsequently minimized K+ leakage (Niu et al. 1995). Therefore, under an alkaline salt treatment, References lower Ca2+ content in krishum was responsible for a lower K/Na. Agarwal, S., K. R. Sairam, G. C. Srivastava, T. Aruna, and Plants have well-developed defence enzymes against C. R. Meena, 2005: Role of ABA, salicylic acid, calcium and ROS including SOD, POD and CAT. SOD is the primary hydrogen peroxide on antioxidant enzymes induction in ) scavenger of O2 and its enzymatic action results in the wheat seedlings. Plant Sci. 169, 559–570. Amtmann, A., and D. Sanders, 1999: Mechanism of Na+ formation of H2O2 and O2. POD and/or CAT then scav- enge the hydrogen peroxide produced by the enzymatic uptake by plant cells. Adv. Bot. Res. 29, 75–112. reactions of SOD (Dionisio-Sese and Tobita 1998, Garratt Arnon, D. I., and D. R. Hoagland, 1940: Crop production in et al. 2002). SOD together with POD and CAT protects artificial culture solutions and in soils with special reference plasma membranes against environmental stresses. A close to factors influencing yields and absorption of inorganic relationship between these enzymes results in plant nutrients. Soil Sci. 50, 463–483. Ashraf, M., 1994: Breeding for salinity tolerance in plants. Crit. resistance to environmental stresses and a more efficient Rev. Plant Sci. 13, 17–42. antioxidative system (Gossett et al. 1994, Sreenivasulu Ashraf, A., and A. Khanum, 1997: Relationship between ion et al. 2000, Bor et al. 2003). Our results found that accumulation and growth in two-spring wheat lines differing SOD, POD and CAT activities were higher in neutral

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