Acta Physiol Plant (2011) 33:2083–2090 DOI 10.1007/s11738-011-0747-3

ORIGINAL PAPER

Salt tolerance analysis of Arabidopsis thaliana NOK2 accession under saline conditions and potassium supply Rym Kaddour • Olfa Baatour • Hela Mahmoudi • Nawel Nasri • Maha Zaghdoudi • Mokhtar Lachaaˆl

Received: 14 December 2010 / Revised: 1 March 2011 / Accepted: 23 March 2011 / Published online: 5 April 2011 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2011

Abstract The response to salt treatment and K? provision of two Arabidopsis thaliana accessions grown for 17 days in the presence of 50 mM NaCl was investigated. Leaf and root dry weight deposition was restricted by salt, more in Col accession than in NOK2 accession. In both accessions, the growth inhibition induced by salinity was associated with a decrease in total leaf surface area, which resulted from diminished leaf number, but not from restriction of individual leaf surface area. Comparing the effects of salt on dry matter production and total leaf surface area revealed large difference between Col and NOK2 for net assimilation rate (the amount of whole plant biomass produced per unit leaf surface area), which was augmented by salt and K? in NOK2 but not in Col. This result, which suggested a better capacity of NOK2 to preserve its photosynthetic machinery against salt stress, was in agreement with the effect of NaCl on photosynthetic pigments. Indeed, salt significantly reduced chlorophyll and carotenoid content in Col leaves but had no impact on NOK2 leaf pigment content. Since K? provision had only marginal effects on these responses to salt stress, leaf mineral unbalance was unlikely. Guaiacol peroxidase activity was augmented by salt treatment in leaves and roots of both accessions. Salinity decreased the catalase

R. Kaddour and O. Baatour have equally participated in the elaboration of the manuscript. Communicated by R. Aroca. R. Kaddour (&)  O. Baatour  H. Mahmoudi  N. Nasri  M. Zaghdoudi  M. Lachaaˆl Physiologie et Biochimie de la Tole´rance au Sel des Plantes, Faculte´ des Sciences de Tunis, Campus Universitaire, 1060 Tunis, Tunisia e-mail: [email protected]

activity in Col leaves and in roots, and increased this activity in NOK2 organs. In conclusion, when aggressed by salt, NOK2 was able (1) to produce more leaves than Col, and (2) to efficiently protect its photosynthetic apparatus, perhaps by developing more efficient antioxidative defense through increased catalase and peroxidase activities. Consequently, the overall photosynthetic activity was higher and more robust to salt aggression in NOK2 than in Col. Keywords Arabidopsis thaliana  NaCl  K?  Growth  Net assimilation rate  Catalase  Guaiacol peroxidase Abbreviations DW Dry weight LSA Leaf surface area NAR Net assimilation rate ROS Reactive oxygen species POD Guaiacol peroxidase

Introduction Salinity is one of the most frequent abiotic stresses confronting worldwide plant agriculture. It is known to adversely affect K? uptake and transport by plants (Schroeder et al. 1994; Amtmann et al. 2006; Shabala and Cuin 2008), and this dysfunction can often be relieved by increased K? supply (Cakmak 2005). Improvement of plant salt tolerance by K? addition has been demonstrated in tomato (Satti and Lopez 1994; Song and Fujiyama 1996), bean (Benlloch et al. 1994), corn (Botella et al. 1997) and sunflower (Delgado and Sanchez-Raya 1999). Using split root experiments with Col accession of Arabidopsis, Attia et al. (2008b) showed that salt-induced

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growth inhibition could be attributed to restricted K? supply to leaves. Ionic and osmotic stresses generated by high salinity may induce secondary oxidative stress and associated damages (Hasegawa et al. 2000; Bartels and Sunkar 2005). Oxidative stress is due to reactive oxygen species (ROS) generated by photosystems and various oxidases (Kawano et al. 2001). Plants have developed both enzymatic and non-enzymatic mechanisms for scavenging these forms. The enzymatic mechanisms, which are mainly represented by superoxide dismutases, catalase and other peroxidases, are acting to minimize the concentration of O2- and H2O2. Furthermore, salinity modifies the level of compounds that can scavenge ROS by reacting directly with them, such as carotenoids (de Pascale et al. 2001). Salt-induced oxidative stress can be alleviated by improving K? nutrition in K-deficient plants (Cakmak 2005). Indeed, a study of the salt response of the whole superoxide dismutase gene complement in Arabidopsis revealed that the changes in SOD transcript abundance accurately reflected the severity of the salt stress mitigated through improved mineral nutrition (Attia et al. 2008a). In a further study (Attia et al. 2009), these authors showed that salt-induced oxidative stress might result from imbalance between light energy input and metabolic energy use when the latter was limited by insufficient K? provision. In Arabidopsis, NOK2 plants are taller than Col plants of the same age, more than twice larger in total leaf surface area, plant biomass, and number of leaves. In a previous study (Kaddour et al. 2009), we observed that in the presence of 50 mM NaCl for 17 days, NOK2 growth was less sensitive to salt than that of Col. Furthermore, increasing external K? concentration from 0.1 to 0.625 or 2.5 mM suppressed the salt-induced inhibition of biomass production in NOK2 but not in Col. In the present study, we examined the role of K? on the salt tolerance of two salt contrasting accessions of Arabidopsis: the salt-sensitive Col accession and the salttolerant NOK2 accession.

Materials and methods Plant material and treatments Seeds of NOK2 and Col accessions provided by the Nottingham Arabidopsis Stock Center (references N1403 and N907, respectively), were grown in pots containing a 1:2 (v:v) mixture of sand and peat, and placed in a culture chamber with a 12 h photoperiod (150 mmol m-2 s-1 PAR). The substrate contained *4.9 mg kg-1 of K? and *4.6 mg kg-1 of Na? per pot. The seedlings were irrigated with distilled water during the first 10 days, then for

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13 days with a Gay and Hauck (1994) nutritive solution (1.0 mM MgSO4; 0.625 mM KH2PO4; 0.5 mM Ca(NO3)2; 1.25 mM NH4NO3; 0.04 lM MnSO4; 0.5 lM ZnSO4; 0.05 lM H3BO3; 0.02 lM MoO3; 3 lM FeEDTA), diluted to the quarter. The first harvest was then performed. The plants were thereafter irrigated with the same nutritive solution modified to contain 0.1 (0.1 mM KH2PO4 ? 0.525 mM NaH2PO4), 0.625 (0.625 mM KH2PO4) or 2.5 mM (0.625 mM KH2PO4 ? 1.875 mM KCl) final K? concentration and 0 or 50 mM NaCl. Seventeen days later, nine plants of each treatment were harvested. Fresh and dry matter of leaves and roots were determined. For each rosette, all the leaves were cut and photographed, and the individual leaf surface areas were measured using OptimasÒ software. Net assimilation rate (NAR, g m-2 days-1) was defined as in Hunt (1990): NAR ¼ D ðDWWP Þ = ðMSL : DtÞ where D DWWP is the increase in the whole plant dry weight during the treatment duration Dt, and MSL is the mean of the total leaf surface area calculated over this period. As the growth kinetics was expected to be exponential rather than linear, MSL was calculated as the logarithmic mean of the total leaf surface area = DSL/D ln(SL) where D stands for the difference between the initial and final harvests. Relative growth rate (RGR, mg mg-1 days-1) was estimated as Kingsbury et al. (1984). RGR ¼ ðln w2  ln w1 Þ = ðt2  t1 Þ where w1 and w2 are dry weights of rosette in milligrams at times t1 and t2 (in days). Pigment quantification Ten millimeter2 diameter discs were cut from fresh leaf samples and incubated in the dark for 72 h at 4°C in acetone 80% (v/v). The absorbance was measured with a UV/ visible spectrophotometer (Jenway PFP7). Absorbance of the acetone extracts measured at 470, 646 and 663 nm was used to calculate concentrations of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids using equations proposed by Lichtenthaler (1988). Extraction of leaf and root proteins Rosette leaves and roots were separately ground in liquid N2, and the obtained powder was resuspended in a 50 mM, pH 7.5 phosphate buffer containing 1 mM EDTA, 1 mM DTT, 5% glycerol and 5% polyvinylpyrrolidone, and centrifuged for 20 min at 15,0009g. Leaf and root soluble protein content were determined in the supernatant

Acta Physiol Plant (2011) 33:2083–2090

Catalase activity from rosette leaves and roots were measured using a modified Chance and Maehly (1955) method. The reaction mixture contained 25 mM K? phosphate buffer pH 7.0, 30 mM H2O2 and enzyme extract. The decomposition of H2O2 was monitored by measuring the decrease in absorbance at 240 nm. Guaiacol peroxidase activity (POD) was assayed according to a method based on Fielding and Hall (1978). The reaction mixture contained 50 mM K? pH 7.0 phosphate, 0.1 mM EDTA, 5 mM H2O2 and 10 mM guaiacol as an electron donor. The increase of absorbance, due to tetraguaiacol formation, was recorded at 470 nm. All enzyme activities were expressed per mg of total soluble proteins. Statistics Data are presented as the mean of nine plants for each treatment. Significant differences between treatments were identified at p = 0.05 level using ANOVA and mean comparison with Duncan’s test (StatisticaÒ).

Rosette biomass (mg DW. plant -1)

Enzyme activity assays

500

0.1 mM K+ 0.625 mM K+ 2.5 mM K+

A 400

B

300

C

200 a

a

a b

100

Col 0 mM NaCl

40

b

b

Col 50 mM NaCl

NOK2 0 mM NaCl

NOK2 50 mM NaCl

B

30

A AB

AB BC C BC

20

a

ab ab

b

10

b

b

0

Results

Col 50 mM NaCl

NOK2 0 mM NaCl

NOK2 50 mM NaCl

C a 0.2

RGR (mg mg -1 d-1)

The impact of different K? supplies on growth was examined both in the presence and in the absence of 50 mM NaCl, which represents a mild salt constraint for Arabidopsis. Leaf biomass was more restricted by salt in Col than in NOK2. In Col, leaf biomass reduction by NaCl 50 mM was 35% (at 0.1 mM K?), 50% (at 0.625 mM K?), and 31% (at 2.5 mM K?). In NOK2, these values were 14, 15 and 0%, respectively (Fig. 1a). Thus, leaf growth inhibition by salt was poorly sensitive to K? in Col, but totally relieved by 2.5 mM K? in NOK2 (Fig. 1a). The variations in the relative growth rate (RGR) showed significant differences between Col and NOK2 when NaCl treatment was considered, but not with potassium addition (Fig. 1c). The RGR was reduced in Col while it remained the same as the control in NOK2. In both accessions, salt inhibition of root biomass was much less pronounced when compared to that of leaves, and was often affected by K? level. Indeed, salt treatment reduced significantly the root biomass at 2.5 mM K? in Col accession and at 0.1 mM in NOK2 accession (Fig. 1b).

A

B

Col 0 mM NaCl

Effects of NaCl and K? on growth, individual and total leaf surface area, leaf number and net assimilation rate

A A

0

Root biomass (mg DW. plant-1)

according to the method of Bradford (1976) using bovine serum albumin (BSA) as the standard.

2085

a

AB

a b

b

A AB

AB AB B

b

0.1

0.0 Col 0 mM NaCl

Col 50 mM NaCl

NOK2 0 mM NaCl

NOK2 50 mM NaCl

Fig. 1 Combined effects of K? concentration and salt on rosette (a) and root growth (b) and on the relative growth rate of rosette (RGR) (c). Col (Colombia-0) and NOK2: accessions. The K? and NaCl treatments were applied to 23-day-old individual plants and lasted 17 days. Means of nine repetitions and confidence intervals (p = 0.05). ANOVA analysis was used and statistically significant mean differences were then identified using Duncan test (letters on the figures). ANOVA analysis was performed separately for each accession. Lower and upper case letters correspond to Col and NOK2 ANOVA analysis, respectively

The individual leaf surface area was insensitive to salt in both accessions, and slightly augmented upon increasing K? supply independently of the presence of salt (Fig. 2a). On the contrary, an important reduction in leaf number

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The two accessions differed in their capacity to accumulate ions in their leaves. When grown on 50 mM NaCl, NOK2 accumulated more K? and much less Na? in its leaves than did Col (Table 1). The increase in leaf K? content is associated to a non-significant decrease of the leaf Na? content in salty NOK2 plants, and had no effect on the leaf Na? content in Col plants (Table 1). This result was traduced by a higher K?/Na? concentration ratio in the more tolerant accession, NOK2 (Kaddour et al. 2009). Effects of NaCl and K? on chlorophyll and carotenoid content in leaves In Col, 50 mM NaCl decreased leaf content in chlorophylls (Chla, Chlb, total Chl) and carotenoids, however, the Chla/ b ratio did not change when compared to the control (Table 2). In NOK2, on the contrary, chlorophyll and carotenoid content were not affected by salt. Independently of salt treatment, increasing K? provision from 0.1 to 2.5 mM, significantly augmented chlorophyll accumulation in leaves of both accessions (Table 2). For both accessions, K? supply had no impact on carotenoid content in salty or control medium. Effects of NaCl and K? on antioxidant enzymatic activities Catalase and POD activities are shown in Tables 3 and 4. Catalase activity tends to decrease by salt both in Col leaves and roots, and to increase only in NOK2 roots. In addition, it increased significantly in leaves of the latter accession. The highest catalase activity was observed for the two accessions at 0.625 mM K?, particularly when the

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0.1 mM K+ 0.625 mM K+ 2.5 mM K+

A

A

3 a

a 2

BC

C

C

ab

BC

AB

b

b ab

1

0 Col 50 mM NaCl

NOK2 0 mM NaCl

NOK2 50 mM NaCl

50

B Rosette leaf number (plant -1)

Effects of NaCl and K? on leaf Na? and K? concentration

4

Col 0 mM NaCl

40

a

A a

a

A A

b

30 c

B

B B

c

20

10

0 Col 0 mM NaCl

120

Total surface area (cm2.plant -1)

occurred in the presence of salt in Col as in NOK2, and K? supply had no effect on this parameter except in Col at 2.5 mM K?, where the salt effect was partially alleviated (Fig. 2b). As a result of these effects of salt on leaf initiation and leaf expansion, the total leaf surface area per plant was larger in NOK2 than in Col, both in the presence and in the absence of salt. (Fig. 2c): total leaf surface area was reduced by 29 and 38% in presence of NaCl 50 mM, respectively, in NOK2 and Col. Furthermore, in both accessions, increasing K? supply limited the detrimental effect of salt on total leaf surface area (Fig. 2c). Finally, the net assimilation rate, which represents the amount of biomass deposited per unit of leaf biomass during the treatment period, was poorly dependent on salt and K? supply in Col (Fig. 3). On the contrary, it tended to increase by both salt and K? supply in NOK2.

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Individual leaf surface area (cm2.leaf -1)

2086

Col 50 mM NaCl

NOK2 0 mM NaCl

NOK2 50 mM NaCl

A

C B B

90 a

CD C

a b

D

60 cd

c

d 30

0 Col 0 mM NaCl

Col 50 mM NaCl

NOK2 0 mM NaCl

NOK2 50 mM NaCl

?

Fig. 2 Combined effects of K concentration and salt on a individual leaf surface area, b leaf number and c total leaf surface area of rosette leaves. Col (Colombia-0) and NOK2 accessions. The K? and NaCl treatments were applied to 23-day-old individual plants and lasted 17 days. Means of nine repetitions and confidence intervals (p = 0.05). ANOVA analysis was used and statistically significant mean differences were then identified using Duncan test (letters on the figures). ANOVA analysis was performed separately for each accession. Lower and upper case letters correspond to Col and NOK2 ANOVA analysis, respectively

medium was added with salt. Furthermore, NaCl treatment tends to increase POD activity in both Col and NOK2 (Tables 3, 4). As for catalase, the highest POD activities in

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2087

0.1 mM K+ 0.625 mM K+ 2.5 mM K+

A A

NAR (g. m-2. d-1.)

700 AB

AB BC

C 500 ab

a ab

ab

ab b

300

100 Col 0 mM NaCl

Col 50 mM NaCl

NOK2 0 mM NaCl

NOK2 50 mM NaCl

Fig. 3 Combined effects of K? concentration and salt on net assimilation rate (NAR). NAR is the amount of whole plant biomass produced per unit leaf surface area. Col (Colombia-0) and NOK2: accessions. The K? and NaCl treatments were applied to 23-day-old individual plants and lasted 17 days. Means of nine repetitions and confidence intervals (p = 0.05). ANOVA analysis was used and statistically significant mean differences were then identified using Duncan test (letters on the figures). ANOVA analysis was performed separately for each accession. Lower and upper case letters correspond to Col and NOK2 ANOVA analysis, respectively

the two accessions were observed in the presence of 0.625 mM K?.

Discussion The biomass produced by the two Arabidopsis accessions, as illustrated by leaf and root dry weight, was more depressed by salt in Col than in NOK2. Growth rate is expected to be positively related with photosynthetic capacity, which itself is determined by the total leaf surface area and the specific photosynthetic activity of leaves. In Col, salt limited strongly the total leaf surface area by almost halving the leaf number, leaf expansion being not significantly modified. A previous study (Kaddour et al. 2009) has shown that leaf thickness was diminished by salt

in Col and augmented in NOK2, but this morphological change was unlikely to have strongly modified leaf specific photosynthetic activity. Indeed, this activity, as estimated from NAR, was poorly sensitive to salt, being unchanged (0.1 and 2.5 mM K?) or diminished at 0.625 mM K?. Thus, the growth reduction in salt-treated Col plants likely resulted principally from restriction in leaf initiation or leaf survival, and to a much lesser extent from limited photosynthesis efficiency. This situation contrasted with that in NOK2. Concerning leaf number and leaf surface area, the difference between the two accessions was essentially quantitative, the leaf number being almost halved by salt in Col, and only reduced by one third in NOK2. NAR was not affected by the salt treatment in the Col accession; therefore, in this genotype photosynthetic machinery is not sensitive to the salt treatment. However, NAR increased by salt treatment in NOK2 accession, what mean that this could be a physiological mechanism to adapt it to the salt condition. In other case, the net photosynthesis rate is more affected by salinity in the salt-sensitive genotypes than in the salt-tolerant ones (Zhao et al. 2007; Lopez-Climent et al. 2008), but not in our experiment. In Col accession, NAR was not affected by salt treatment; however, leaf chlorophyll content was significantly decreased. Some studies showed no positive correlation between the NAR and chlorophyll content values. For example, in several wheat genotypes, the NAR values were decreased by salt, whereas chlorophyll content was genotype-dependent (El-Hendawy et al. 2005). The status of leaf Na? is another characteristic that discriminates the two Arabidopsis accessions. NOK2 accession is a low Na?-accumulator and Col is a high Na?-accumulator and this could explain, among other physiological mechanism, the contrasting salt tolerance differences observed between these two accessions. In addition to the Na? accumulation, in the Table 3, differences in the antioxidant enzymes system are also observed between accessions such that in the more sensitive accession, the salt treatment tends to inhibit this system, but in

Table 1 Combined effects of K? concentration and salt on ion content of rosette leaves Accession

ion

No NaCl

50 mM NaCl added ?

0.1 mM K

0.625 mm K

?

?

2.5 mM K

0.1 mM K?

0.625 mm K?

2.5 mM K?

Col

K?

554b

818a

904a

304c

334c

471bc

NOK2

K?

699C

1,060B

1,252A

436D

622CD

914B

Col

Na?

317c

436c

577c

4034b

4,664a

3,889b

NOK2

Na?

168B

245B

255B

2,052A

1,978A

1,534A

Contents are expressed as lmol g-1 DW. The treatments were applied to 23-day-old individual plants and lasted 17 days. Means of nine repetitions. Low, medium, and high K? refer to K? concentration in the irrigation medium: 0.1, 0.625 and 2.5 mM, respectively. Col (Colombia-0) and NOK2: accessions. Means of nine plants and confidence intervals at p = 0.05. For each ion, values sharing a same letter are not significantly different at p = 0.05 (ANOVA and Duncan’s test for mean comparison)

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Table 2 Effect of salt and K? concentration on pigment content in leaves of two Arabidopsis thaliana accessions (Col and NOK2) 0.1 mM K?

0.625 mM K?

2.5 mM K?

Control

Salt

Control

Salt

Control

Salt

Chla (mg g-1 leaf DW)

0.38b

0.18d

0.45ab

0.25cd

0.48a

0.29c

Chlb (mg g-1 leaf DW)

0.18b

0.09c

0.24a

0.13c

0.23a

0.13bc

Col accession

Chl tot (mg g

-1

0.55b

0.30d

0.74a

0.40 cd

0.77a

0.45bc

Chla/b

leaf DW)

2.21a

1.96a

1.94a

1.97a

2.05a

2.23a

Car (mg g-1 leaf DW)

0.30a

0.16b

0.27ab

0.17b

0.29a

0.18b

0.45BC

0.40C

0.53AB

0.50AB

0.56A

0.53AB

NOK2 accession Chla (mg g-1 leaf DW) Chlb (mg g

-1

0.23BC

0.22C

0.27AB

0.26ABC

0.28A

0.28AB

Chl tot (mg g-1 leaf DW)

leaf DW)

0.73BC

0.67C

0.86AB

0.82AB

0.90A

0.86AB

Chla/b

1.93A

1.88A

1.97A

1.92A

1.98A

1.91A

Car (mg g-1 leaf DW)

0.28A

0.28A

0.26A

0.29A

0.31A

0.29A

Control no NaCl added, Salt the medium was supplemented with 50 mM NaCl, Chl chlorophyll, Car carotenoids The seedlings were treated for 17 days with different potassium and NaCl concentrations. Means of three replicates. Significant differences between treatments were determined using ANOVA and mean comparison with Duncan post hoc test (StatisticaÒ) at p = 0.05 level. ANOVA analysis was performed separately for each accession

Table 3 Protein content and activities of antioxidant enzymes in Col and NOK2 leaves 0.1 mM K?

0.625 mM K?

2.5 mM K?

Control

Salt

Control

Salt

Control

Salt

Protein (mg g-1 DW-1)

1.4a

1.38a

1.34a

1.36

1.54

1.32a

Catalase (U mg-1 protein)

2.95a

1.40b

2.71a

2.09ab

1.97ab

1.74ab

POD (U mg-1 protein)

4.60c

5.70bc

6.74b

9.95a

4.42c

6.10bc

Protein (mg g-1 DW-1)

1.46AB

1.23B

1.80A

1.83A

1.46AB

1.59AB

Catalase (U mg-1 protein) POD (U mg-1 protein)

1.99C 4.00D

3.29B 5.81CD

3.65B 6.09BC

5.97A 11.4A

2.01C 5.54CD

4.00B 7.94B

Col accession

NOK2 accession

Control no NaCl added, Salt the medium was supplemented with 50 mM NaCl, POD guaiacol peroxidase The seedlings were treated for 17 days with different potassium and NaCl concentrations. Means of three replicates. Significant differences between treatments were determined using ANOVA and mean comparison with Duncan post hoc test (StatisticaÒ) at p = 0.05 level. ANOVA analysis was performed separately for each accession

the NOK2 accession, the salt induced an increase of the POD and catalase activities. Potassium fulfills important functions, and when it is lacking, growth is disrupted as a consequence (Marchner 1995; Amtmann et al. 2006). Addition of K? improves salt tolerance of tomato (Satti and Lopez 1994; Song and Fujiyama 1996), corn (Botella et al. 1997), sunflower (Delgado and Sanchez-Raya 1999) and bean (Benlloch et al. 1994). Our results suggest that K? provision improved NOK2 salt tolerance. Indeed, enrichment of the medium with K? increased only NOK2 rosette biomass and the K?/Na? ratio. The NAR values were also slightly

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increased. It has been shown that restriction of K? uptake is a major component of salt stress in Arabidopsis (Attia et al. 2008b). For Col on the contrary, K? did not seem to be limitant even at 0.1 mM, perhaps because the growth rate of this accession was much lower than that of NOK2. Salinized NOK2 plants showed, in all K? treatments, the similar leaf Na? concentration, but the leaf K? concentration increased with increasing the K? concentration in the root medium, therefore, maintaining intracellular K? and Na? homeostasis to preserve a high K/Na ratio had a key important in the different response to the salt under different K? conditions. In addition, maintaining a highest

Acta Physiol Plant (2011) 33:2083–2090

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Table 4 Protein content and activities of antioxidant enzymes in Col and NOK2 roots 0.1 mMK? Control

0.625 mM K? Salt

Control

2.5 mM K? Salt

Control

Salt

Col accession Protein (mg g-1 DW-1)

0.35b

0.23b

0.25b

0.71

0.43ab

0.30b

Catalase (U mg-1 protein)

1.18bc

0.73c

3.00a

3.14a

2.94a

1.84b

POD (U mg-1 protein)

13.5c

16.8bc

15.9bc

25.4a

16.1bc

19.1b

NOK2 accession Protein (mg g-1 DW-1)

0.22BC

0.34BC

0.20C

0.52AB

0.28BC

0.69A

Catalase (U mg-1 protein) POD (U mg-1 protein)

2.86C 14.0D

3.14BC 16.2CD

3.15BC 14.5CD

5.72A 27.5A

3.21BC 18.8BC

4.16B 20.7B

Control no NaCl added, Salt the medium was supplemented with 50 mM NaCl, POD guaiacol peroxidase The seedlings were treated for 17 days with different potassium and NaCl concentrations. Means of three replicates. Significant differences between treatments were determined using ANOVA and mean comparison with Duncan post hoc test (StatisticaÒ) at p = 0.05 level. ANOVA analysis was performed separately for each accession

leaf K? concentration is critical for plant growth and salt tolerance of many plant species when grown under saline habitats (Carden et al. 2003; Golldack et al. 2003; Ghars et al. 2008). Salt decreased chlorophyll and carotenoid content in Col, but not in NOK2. The decrease in chlorophyll content is considered as an indicator of sensitivity to salt stress (Singh and Dubey 1995). In mungbean, for instance, steady levels of carotenoids are strongly correlated to its tolerance of salt stress (Wahid et al. 2004), and in sugarcane, lesser reduction in chlorophylls associated with steady levels of carotenoids seems to be a strategy for salt tolerance (Kanhaiya 1996). Thus, salt stress seemed more acute in Col leaves than in NOK2 ones. However, the very limited effect of K? provision on pigment contents in both accessions suggests that perturbation of leaf K? nutrition was not involved in the stress affecting the pigments. Induction of ROS scavenging activities such as catalase and peroxidases is generally considered as a proxy for oxidative stress (Attia et al. 2009). Catalase activity was decreased by salt in Col leaves and roots, whereas it was increased in NOK2 organs. In the latter accession, this response might participate in protecting leaf biochemical structures against oxidative damages and minimizing salt sensitivity of the photosynthetic machinery. Similarly, guaiacol peroxidase activity differed between the two accessions, being increased by NaCl more in NOK2 than in Col. Thus, it is reasonable to infer that both accessions suffered from oxidative stress when submitted to salt, and that NOK2 was more efficient than Col in developing antioxidative defense through increased catalase and peroxidase activities. Such correlation between the magnitude of the antioxidative response and the relative salt tolerance has been observed in several plant species when varieties

(Sairam et al. 2000, 2001) or related species (Mittova et al. 2002) were compared. The observed increase in catalase and peroxidase activity upon K? addition is difficult to explain. Several examples of such effect are known, in various plants and conditions. This response has been described in Mg2? deficient rice leaves (Ding et al. 2008). In cucumber grown on soil, increase in catalase activity by K? supply has been described by Moreno et al. (2003). Mineral disequilibrium aggravated by K? supply increase was invoked to explain these effects of K?, but no mechanistic explanation is available. The expression of defense systems is a cellular process, which necessitates that the cell machinery is not damaged. In our experiments, it is possible that at the lowest K? level, salt aggression associated with K? deficiency resulted in deteriorated physiological conditions limiting the deployment of antioxidative defense. In this case, provision of K? to salt stressed plants would have restored the expression of these systems. Similar mechanism have been proposed to rationalize the complex pattern observed for the expression of the SOD gene family in Arabidopsis (Attia et al. 2008a): the changes in SOD transcript abundance in leaves of salt-treated plants seemed to reflect adaptative gene induction superimposed to apparent repression, originating from general damages to the biochemical machinery. In conclusion, the difference in salt sensitivity between Col and NOK2 seems to result from differential aptitude to protect the leaves against salt stress, including the resulting oxidative stress. When aggressed by salt, NOK2 was able (1) to produce more leaves than Col, and (2) to efficiently protect its photosynthetic apparatus, perhaps by developing more efficient antioxidative defense through increased catalase and peroxidase activities. Consequently, the photosynthetic activity was higher and more robust to salt aggression in NOK2 than in Col.

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