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The K/Na replacement and function of antioxidantdefence system in sugar beet (Beta vulgaris L.)cultivarsDr Roghieh Hajiboland a & Arshad Joudmand aa Plant Science Department, University of Tabriz, 29 Bahman Ave, 51666, Tabriz, IranPublished online: 22 Jul 2009.

To cite this article: Dr Roghieh Hajiboland & Arshad Joudmand (2009) The K/Na replacement and function of antioxidantdefence system in sugar beet (Beta vulgaris L.) cultivars, Acta Agriculturae Scandinavica, Section B - Soil & Plant Science,59:3, 246-259, DOI: 10.1080/09064710802029544

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ORIGINAL ARTICLE

The K/Na replacement and function of antioxidant defence system insugar beet (Beta vulgaris L.) cultivars

ROGHIEH HAJIBOLAND & ARSHAD JOUDMAND

Plant Science Department, University of Tabriz, 29 Bahman Ave, 51666, Tabriz, Iran

AbstractSalinity is one of the major constrains restricting crop productivity. Sugar beet is one crop species grown mainly onsalinity-affected soils. In a preliminary experiment in this work, the effect of different NaCl concentrations was studied onthe growth of six cultivars of sugar beet (Beta vulgaris L.) in hydroponic medium. After selection of two contrastingcultivars, growth, sodium accumulation and distribution among leaf fractions as well as the functional significance of theantioxidant defence system under mild (50 mM) and severe salinity (200 mM) were investigated. NaCl treatment exertedeither a stimulatory or an inhibitory effect on shoot growth, depending on concentration and cultivar. Higher growthimprovement under mild salinity was observed in cultivar IC simultaneous with higher tolerance to severe salinetreatment. Although two tested cultivars did not differ in Na concentration of leaves, its distribution among leaf fractions(apoplasmic fluid, cell wall, and cell sap) responded differentially to salinity treatment. Under mild salinity, proportionalNa in cell sap of cultivar IC was greater than that of cultivar 7233, leading to greater replacement of K by Na in theformer cultivar. In leaves of 7233, in contrast, proline was the major osmoticum. Activity of H2O2-scavenging enzymes(APX, CAT, and POD) was induced by salinity, keeping H2O2 concentration low under mild but not severe salinity.Shoot and root concentration of superoxide radicals was related to differential response of cultivars to salinity, i.e. IC wasmuch more protected against superoxide radicals because of higher SOD activity than was 7233. Activity of nitratereductase was reduced by both salinity levels and caused reduction of total free amino acids and protein concentrations.Results suggested that the main cause of cultivar difference is higher replacement of K by Na and allocation of more Nato the symplasm under mild salinity, and an indigenously higher protection against superoxide radicals under severesalinity.

Keywords: Ascorbate peroxidase, catalase, mild salinity, nitrate reductase activity, peroxidase, superoxide radicals.

Introduction

Worldwide about 33% of the irrigated lands are

affected by salinity. Saline soils are abundant parti-

cularly in semiarid and arid regions, where the

amount of rainfall is insufficient for substantial

leaching. Salinity can severely limit crop yield,

especially in the most productive areas of the world

(Pitman & Lauchli, 2004).

Under salt stress, plants have evolved complex

mechanisms allowing for adaptation to osmotic and

ionic stress caused by high salinity. In plant species

in which salt inclusion is the predominant strategy

and osmotic adjustment is achieved by the accu-

mulation of salts (mainly NaCl) in the leaf tissue,

e.g. members of the Chenopodiaceae, a strict

compartmentation of Na and Cl ions occurs in

the vacuoles and, accordingly, nontoxic or compa-

tible organic solutes such as glycine betaine and

proline accumulate in the cytoplasm and chloro-

plasts for osmotic adjustment (Cushman, 2001;

Rhodes et al., 2004).

Salt tolerance in plants has generally been empha-

sized in regulation of ionic homeostasis and osmotic

adjustment (Leidi & Saiz, 1997; Rhodes et al.,

2004). Some authors have shown that, like other

abiotic stresses, salinity also induces oxidative stress

in plants (Bartoz, 1997; Fadzilla et al., 1997; Santos

et al., 2001). In pea leaves sodium chloride toxicity is

associated with higher generation of hydrogen per-

oxide (Gomez et al., 2004). One of the primary

effects of reactive oxygen species (ROS) and their

products in cells is the peroxidation of membranes,

Correspondence: Dr Roghieh Hajiboland, University of Tabriz, Plant Science Department, 29 Bahman Ave, 51666, Tabriz, Iran. Email: ehsan@tabrizu.ac.ir

Acta Agriculturae Scandinavica Section B � Soil and Plant Science, 2009; 59: 246�259

(Received 22 December 2007; accepted 3 March 2008)

ISSN 0906-4710 print/ISSN 1651-1913 online # 2009 Taylor & Francis

DOI: 10.1080/09064710802029544

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which leads to leakage of low-molecular-weight

solutes, particularly K� (Santos et al., 2001). Plants

have evolved various protective mechanisms to

eliminate or reduce ROS, which are effective at

different levels of stress-induced deterioration. The

enzymatic antioxidant system, which is one of the

protective mechanisms, including superoxide dismu-

tase (SOD), is located in various cell compartments

and catalyses the disproportion of two O2.� radicals

to H2O2 and O2. H2O2 is eliminated by various

antioxidant enzymes such as catalase (CAT) and

peroxidases (POD), which convert H2O2 into water

(Creissen & Mullineaux, 2002). Ascorbate perox-

idase (APX) eliminates peroxides by converting

ascorbic acid into dehydroascorbate (Ben-Hayyim

et al., 1999). Ascorbate peroxidase and glutathione

reductase (GR) are important components of the

ascorbate�glutathione cycle responsible for the re-

moval of H2O2 in different cellular compartments

(Jimenez et al., 1997). In addition to the involve-

ment of proline in tolerance to water deficiency and

salt stress (Santos et al., 2001), it plays a significant

role in scavenging of hydroxyl radicals (Alia &

Mohanty, 1997).

It could be speculated that the antioxidant defence

capacity of plants could not only be involved in

protection of plants against stress-induced ROS

production, but could also explain differences

among tolerant and susceptible species/genotypes

to stress. The functional significance of antioxidant

defence capacity in adaptation of plants to stressful

conditions is well documented under heavy metal

toxicity (Briat, 2002), other stresses such as higher

light intensity and UV (Apel & Hirt, 2004), as well

as in the interpretation of the beneficial effects of

trace concentrations of nonnutritional elements such

as Se (Hartikainen et al., 2000).

Plant species differ greatly in their growth re-

sponse to salinity. Although sugar beet is sensitive to

elevated salinity at the germination and early seed-

ling phases of development, established plants show

a high osmotic adjustment and accumulation of

proline, glycine betaine, and inorganic ions under

salt stress (Ghoulam et al., 2002).

In terms of response to NaCl, plants were

classified into four major groups, including plant

species with growth stimulation at low salinity due to

the presence of Na as a beneficial element (Marsch-

ner, 1995). A few crop species are classified in this

group, one important example being sugar beet.

The main sugar beet-growing areas in NW Iran

are affected by salinity. This problem may be a

serious handicap for the cultivation and production

of this agricultural crop. To solve this problem, more

tolerant sugar beet varieties must be selected and

recommended for the saline areas. The genetic

variability within a species is not only a valuable

tool for studying mechanisms of salt tolerance

but also is an important basis for screening and

breeding for higher salt tolerance (Gorham & Jones,

2004). Accurate selection requires an understanding

of the mechanisms involved in salt tolerance in this

species.

The importance of osmotic adjustment strategies

in salt tolerance has been extensively investigated in

various plant species. Moreover, involvement of

antioxidant capacity in salinity tolerance has been

suggested by some authors (Meloni et al., 2003).

However, differential response of cultivars to the

growth-improving effect of Na has not attracted

enough attention, either considering the contribut-

ing role of the antioxidant defence system or in terms

of the role of Na in osmotic adjustment mechanisms.

We hypothesized that increased activity of the

antioxidant enzymes SOD, POD, and GR not only

contributes to the protection of tolerant cultivars

from severe salt stress, but also can explain the

beneficial effect of mild salinity in some cultivars.

Therefore, the aim of this work was to evaluate the

effect of salt stress on the antioxidant capacity of

plants and its significance in determining plants’

response to salinity in comparison with the impor-

tance of strategies for osmotic adjustment, Na/K

replacement, and Na fractionation between apo-

plasm and symplasm.

Materials and methods

In a preliminary experiment, six cultivars of sugar

beet (Beta vulgaris L.), namely IC, 7233, 41-RT,

276, Hybrid, and Rasoul, were tested for their

salinity response (up to 200 mM) in hydroponic

medium. Thereafter, two contrasting cultivars (IC

and 7233) were selected for further experiments.

Seeds were provided by the Research Center for

Sugar Beet, West Azerbaijan Province, Iran.

Plant culture and treatments

The experiments were conducted in a growth cham-

ber with a temperature regime of 25 8C/18 8C day/

night, relative humidity of 70/80%, and 14 h/10 h

light/dark period under a photosynthetic photon flux

density of 400�450 mmol m�2 s�1 supplied by

fluorescent lamps. Surface-sterilized seeds were ger-

minated in the dark on filter paper, moistened with

distilled water and CaSO4 at 0.05 mM. After

germination, young seedlings were transferred to

the light and allowed to grow for another two days.

Seven-day-old seedlings were transferred to hydro-

ponic culture in a plastic container with 10 L of half-

strength nutrient solution (Hoagland & Arnon,

Responses of sugar beet cultivars to salinity 247

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1950) and pre-cultured for one week. Thereafter,

plants with uniform size were transferred to

1.2 L dark plastic pots with full-strength nutrient

solution; one plant was cultured in each pot. Salinity

treatments were started for 14-day-old plants, and

consisted of three, four, or five levels of NaCl

(depending on experiment and measurements) at 0

(control), 25, 50, 100, and 200 mM. Nutrient

solutions were completely changed every 5 days;

pH was 6.0 and adjusted every day.

Harvest

In the first experiment with six cultivars, plants were

treated for 21 days. However, it was noted that any

difference among cultivars in response to mild

salinity was gradually diminished from the third

week of treatment. Therefore, in further experiments

with two contrasting cultivars, plants were treated

only for 14 days.

At harvest, shoot, root, and hypocotyls were

separated, washed with distilled water, and blotted

dry. After drying at 70 8C for 48 h in an oven, the dry

weight of samples was determined. Hypocotyl and

root were weighed and analysed separately; however,

because no differential response was observed be-

tween these components, they were considered as

underground organs and are designated as root in

the Results section. For determination of Na, K, and

Ca content, oven-dried samples were ashed in a

muffle furnace at 550 8C for 8 h, dissolved in HCl,

and made up to volume by distilled water (Jaiswal,

2004). The concentration of elements was deter-

mined by flame photometry (JENWAY, PFP7).

Other groups of plants were used for chlorophyll

determination, fractionation experiment, and en-

zymes and metabolites assays.

For determination of chlorophyll concentration,

third leaves immediately after harvest were used for

extraction of chlorophyll by N,N-dimethylforma-

mide according to Moran (1982).

Isolation of apoplasmic fluid and cell sap

Expanded leaves were harvested by cutting their

petioles with a razor blade at the base of the leaf

lamina. After determination of fresh weight, leaves

were arranged all with cut ends oriented in the same

direction on a plastic foil, placed in a centrifugal

tube, and centrifuged for 15 min at 5000 g. The

volume of collected apoplasmic fluid was about 100

mL, which was made up to 5 mL before determina-

tion of Na concentration. After centrifuging, leaves

were frozen at �20 8C for 24 h to rupture the cells.

Cell sap was prepared by squeezing the leaves after

thawing (Dannel et al., 1995).

Measurement of K� leakage from tissues

After being washed with distilled water for 1 min,

either the whole root system of intact plants or leaf

disks (1�1 cm) were transferred to the loading

solution containing 0.5 mM CaSO4, 0.1 mM KCl,

and 1.5 mM MES (2-[N-morpholino]ethanesulfo-

nate) at pH 6.0. After pre-incubation, the plants

were transferred to 100 mL of fresh nutrient solution

and the experiment was started (t�0). The solution

was aerated and kept at 22 8C using water-bath. The

K� concentration of the 2 mL aliquots, which were

taken every 20 min for 2 h, was determined by flame

photometry (De Vos et al., 1989).

Relative water content

Leaf relative water content (RWC) was measured in

fully expanded leaves of three plants per replicate.

Five leaf disks of 10 mm diameter were excised from

the interveinal areas of each plant. For each repli-

cate, 20 discs were pooled and their fresh weight

(FW) was determined. They were floated on distilled

water in Petri dishes for 4 h to regain turgidity, then

blotted dry gently on filter paper and re-weighed

(turgidity weight TW). The samples were dried at 80

8C for 24 h to determine the dry weight (DW). Tests

showed that complete hydration of the leaf disks

occurred within 4 h. RWC was defined as in

Equation (1) (Ghoulam et al., 2002):

RWC (%)�[(FW�DW)=(TW�DW)] �100 (1)

Determination of enzyme activity

Another group of plants was used for assay of

enzymes and metabolites. Fresh leaf samples were

used for enzyme extraction and measurement of

protein and metabolites. Samples were ground in the

presence of liquid nitrogen using a mortar and

pestle. Each enzyme assay was tested for linearity

between the volume of crude extract and the

measured activity.

Ascorbate peroxidase. The enzyme was extracted in 50

mM phosphate buffer (pH 7.0). The activity of

ascorbate peroxidase (APX, EC 1.11.1.11) was

measured using a modified method of Boominathan

and Doran (2002). The reaction mixture consisted

of 50 mM sodium phosphate buffer (NaH2PH4/

Na2HPO4) at pH 7.0 containing 0.2 mM ethylene-

diaminetetraacetic acid (EDTA), 0.5 mM ascorbic

acid (Sigma), 50 mg of bovine serum albumin (BSA)

(Sigma), and crude enzyme extract. The reaction

was started by addition of H2O2 at a final concen-

tration of 0.1 mM. Oxidation of ascorbic acid as a

248 R. Hajiboland & A. Joudmand

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decrease in absorbance at 290 nm was followed

2 min after starting the reaction. The enzyme activity

was calculated using an absorbance coefficient

for ascorbic acid of 2.8 mM�1 cm�1. One unit of

APX oxidizes ascorbic acid at a rate of 1 mmol min�1

at 25 8C.

Catalase

Catalase (CAT, EC 1.11.1.6) activity was assayed

spectrophotometrically by monitoring the decrease

in absorbance of H2O2 at 240 nm (Luck, 1962). The

enzyme was extracted in 50 mM phosphate buffer

(pH 7.0). The assay solution contained 50 mM

phosphate buffer and 10 mM H2O2. The reaction

was started by addition of an enzyme aliquot to the

reaction mixture and the change in absorbance was

followed 2 min after starting the reaction. Unit

activity was taken as the amount of enzyme which

decomposes 1 M of H2O2 in one min.

Peroxidase

Peroxidase (POD, EC 1.11.1.7) activity was deter-

mined using the guaiacol test (Chance & Maehly,

1955). The enzyme was extracted by 10 mM

phosphate buffer (pH 7.0) and assayed in a solution

contained 10 mM phosphate buffer, 5 mM H2O2,

and 4 mM guaiacol. The reaction was started by

addition of the enzyme extract at 25 8C and was

followed 2 min after starting the reaction. The

enzyme unit was calculated as the amount of enzyme

protein required for the formation of 1 mM tetra-

guaiacol in 1 min.

Superoxide dismutase. Total superoxide dismutase

(SOD, EC 1.15.1.1) activity was determined

according to the method of Giannopolitis and

Ries (1977). The enzyme was extracted in 25

mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesul-

fonate (HEPES) (pH 7.8) and 0.1 mM EDTA, and

the homogenate was centrifuged at 15 000 g for 15

min. Test tubes containing 25 mL of enzyme

extract, 25 mL extraction buffer, and 450 mL of

the reaction mixture were incubated in a growth

chamber at 22 8C and at a light intensity of 400

mmol m�2 s�1. The reaction buffer contained 25

mM HEPES (pH 7.6), 0.1 mM EDTA, 50 mM

Na2CO3 (pH 10.2), 12 mM L-methionine, 75 mM

nitroblue tetrazolium (NBT), and 1 mM riboflavin.

The reaction was started by removing a dark plastic

foil from the surface of samples and continued for

10 min. One unit of SOD was defined as the

amount of enzyme required to induce a 50%

inhibition of NBT reduction as measured at 560

nm, compared with control samples without en-

zyme aliquot.

Glutathione reductase. The enzyme was extracted in

50 mM phosphate buffer (pH 7.0) containing 5 mM

EDTA and 2% (w/v) of insoluble poly(vinylpyrroli-

done) (PVP). The extract was centrifuged at 15 000

g in 4 8C for 20 min. The activity of glutathione

reductase (GR, EC 1.6.4.2) was assayed by follow-

ing the oxidation of nicotinamide�adenine dinucleo-

tide phosphate (reduced form) (NADPH) at 340 nm

(extinction coefficient 6.2 mM�1 cm�1) as de-

scribed by Foyer and Halliwell (1976). The reaction

mixture contained 100 mM 2-amino-2-(hydroxy-

methyl)propane-1,3-diol hydrochloride (Tris-HCl)

(pH 7.8), 2.0 mM EDTA, 0.05 mM NADPH, 0.5

mM oxidized glutathione (GSSG), and 50 mL of

enzyme extract at 25 8C. One unit of enzyme activity

was calculated as the amount of enzyme protein

required for oxidation of one mM NADPH in 1 min.

Determination of oxidants and antioxidants

Hydrogen peroxide. The concentration of H2O2 was

determined using methods described by Patterson et

al. (1984). 1�1.5 g of leaf was homogenized with 0.2

g of activated charcoal (Sigma) and 5 mL of 5% w/v

trichloroacetic acid (TCA) in an ice-bath using a

pre-chilled mortar and pestle. The homogenates

were filtered through four layers of cheesecloth and

centrifuged at 14 000 g for 15 min at 4 8C. The

supernatant was then filtered through a 0.45 mm

filter (Millipore). The colorimetric reagent was a 1:1

v/v mixture of 0.6 mM 4-(2-pyridylazo)resorcinol

(disodium salt) and 0.6 mM potassium titanium

oxalate. To a known volume of supernatant, 1 mL of

colorimetric reagent was added and the mixture was

incubated at 45 8C on a heating plate for 60 min.

The absorbance was measured at 508 nm against a

reference solution containing 50 mL of 50% w/v

TCA and 1.95 mL of 100 mM potassium phosphate

buffer (KH2PH4/K2HPO4) at pH 8.4. The concen-

tration of H2O2 was determined from a standard

curve.

Superoxide radical

The assay of NADPH-dependent O2.� generation

was carried out according to the method of Bielski et

al. (1980) by measuring the rate of SOD-inhibitable

NBT reduction. The tissue extracts were prepared in

50 mM Tris-HCl (pH 7.4) and 250 mM sucrose.

Reaction mixtures in the �SOD (sample) cuvettes

contained Tris-HCl (pH 7.4) and 0.1 mM NBT.

The reaction mixture in the �SOD (reference)

Responses of sugar beet cultivars to salinity 249

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cuvette was identical to that in the �SOD cuvette

except for the addition of 50 Unit mL�1 superoxide

dismutase (Sigma). The reaction was started by the

addition of 0.1 mM NADPH at 25 8C. NBT is

rapidly converted into monoformazan by two mole-

cules of O2.�; monoformazan concentration (and

therefore O2.�concentrations) was detected spectro-

photometrically at 530 nm after 15 min. The SOD-

inhibitable component of monoformazan formation

was obtained by subtracting the absorbance for �SOD (total) from that of �SOD (noninhibitable

by SOD) reactions, which was performed automati-

cally by double-beam spectrophotometer (Specord

200, Analytical Jena, Germany) using an extinction

coefficient of 12.8 mM�1 cm�1.

Malondialdehyde. Lipid peroxidation was estimated

from the amount of malondialdehyde (MDA)

formed in a reaction mixture (Heath & Packer,

1968). Leaf tissues were homogenized (1:5) in

0.1% w/v TCA. The homogenate was centrifuged

at 10 000 g for 5 min. To 1 mL of the supernatant, 4

mL of 20% w/v TCA containing 0.5% w/v thiobar-

bituric acid (Sigma) was added. The solution was

heated at 95 8C for 30 min and then quickly cooled

on ice. The mixture was centrifuged at 10 000 g for

15 min and the absorbance measured at 532 nm.

MDA levels were calculated from a 1,1,3,3-tetra-

ethoxypropane (Sigma) standard curve (Boomi-

nathan & Doran, 2002).

Proline concentration. Proline was extracted and its

concentration determined by the method of Bates et

al. (1973). Leaf tissues were homogenized with 3%

sulfosalicylic acid and the homogenate was centri-

fuged at 3000 g for 20 min. The supernatant was

treated with acetic acid and acid ninhydrin, boiled

for 1 h, and then absorbance at 520 nm was

determined. Proline (Sigma) was used for produc-

tion of a standard curve.

Determination of protein, total amino acids, and activity

of NR

Total protein concentration. Soluble proteins were

determined as described by Bradford (1976) using

a commercial reagent (Sigma) and BSA (Merck) as

standard.

Total amino acids. Content of total free a-amino acids

was assayed using a ninhydrin colorimetric method

(Hwang & Ederer, 1975). Leaf tissues were homo-

genized using ice-cold 50 mM phosphate buffer (pH

6.8). The homogenate was centrifuged at 18 000 g

for 20 min. Ninhydrin reagent (1:5 diluted solution

of 350 mg in 100 mL of ethanol) was added to the

sample solution and after gentle stirring the mixture

was incubated for 4�7 min at 80�100 8C in a water-

bath. After cooling of the mixture to room tempera-

ture in a water-bath, the absorbance was recorded at

570 nm. Glycine was used for production of a

standard curve.

Nitrate reductase activity. In vivo nitrate reductase

(NR, E.C. 1.6.6.1) activity was determined using the

method described by Jaworski (1971). Leaf blades

and root samples were cut into 5 mm sections and

placed in incubation buffer (100 mg tissue for 10 mL

of buffer) containing 50 mM K-phosphate buffer

(pH 7.5), 100 mM KNO3, and 1.5% 1-propanol.

The samples were infiltrated using a vacuum (0.8

bar). After 5 min, the vacuum was released and the

samples were incubated at 30 8C in darkness for 1 h

then placed in a boiling water-bath to stop the NR

activity. The resulting NO2.� concentration was

determined spectrophotometrically at 540 nm in a

reaction mixture containing 2 mL of extract, 2 mL of

1% (w/v) sulfanilamide in 1.5 M HCl, and 2 mL of

0.02% (w/v) N-NEDA (naphthylethylenediamine

dihydrochloride) in 0.2 M HCl. NR activity was

calculated from a standard curve established with

NaNO2 concentrations and expressed in produced

mmol NO2.� h�1 g�1 FW.

Experiments were undertaken in complete rando-

mized block design with 4 replications. Statistical

analyses were carried out using Sigma Stat (3.02)

with Tukey test (pB0.05).

Results

Plant growth

In the first experiment, 21 days’ treatment with

different NaCl concentrations caused significant

changes in shoot and root growth mainly under an

NaCl concentration of 100 mM and higher. Sig-

nificant or slight reduction of shoot growth by 50

mM NaCl was observed in cultivars 7233, 276,

Hybrid, and Rasoul. In other tested cultivars,

including IC and 41-RT, rather a slight increase of

shoot growth was observed. According to this

experiment, IC was the most tolerant cultivar to

higher salinity treatment (200 mM) with only 36%

reduction of shoot DW. This cultivar showed also

higher stimulation (though insignificant in this

experiment) of growth under mild (50 mM NaCl)

salinity (Table I).

250 R. Hajiboland & A. Joudmand

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In the experiment using two selected cultivars, dry

matter production of shoot and root decreased

significantly at higher concentrations of NaCl (100

and 200 mM). Growth inhibition of shoot due to

NaCl salinity at 200 mM was higher in 7233 (61% at

200 mM NaCl) than in IC (32%). A similar

difference between two cultivars was observed for

root dry weight: reduction of root biomass with 200

mM NaCl was about 75% and 59% for 7233 and

IC, respectively (Figure 1).

In contrast to higher NaCl salinity level, under

mild salinity (25 and 50 mM) plant growth was

stimulated slightly or significant. Shoot dry weight of

IC increased in response to 50 mM NaCl up to 82%;

the corresponding value for 7233 was only 48%. Leaf

area in IC increased up to 20% at 50 mM NaCl

treatment, while in 7233 it decreased by 20%

following the same treatment. Other growth para-

meters such as root and hypocotyl dry weight did not

respond positively to mild salinity, with the exception

of root DW of IC treated with 50 mM NaCl (Figure

1).Concentration of chlorophyll increased only

slightly with mild salinity and was further diminished

with increasing NaCl treatment (Figure 1).

Concentration of Na, K, and Ca

Concentration of Na in both shoot and root in-

creased with increasing salinity level and tested

cultivars did not differ considerably in the Na

accumulation either in shoot or root. Potassium

concentration was not affected by mild salinity (50

mM) in root. Shoot K concentration remained

unchanged in 7233 at mild salinity treatment, while

it decreased significantly in IC. However, higher

salinity (200 mM) decreased K concentration; this

reduction in roots of 7233 was higher (44%) than in

IC (26%). Concentration of Ca increased in ten-

dency or significantly in shoot and roots of NaCl-

treated plants. However, the increase in Ca content

of shoots in 7233 was only 63% compared with a

121% increase in Ca concentration of IC treated

with 200 mM NaCl (Table II).

Leaf Na concentration on an FW basis was not

different between two tested cultivars (Table III).

However, distribution of Na between apoplasmic

fluid, cell sap, and residual fraction as well as their

change in response to increasing salinity level dif-

fered between cultivars. In 7233, the proportional

Na concentration in cell sap was higher in control

plants and decreased with increasing salinity treat-

ment. In contrast, in IC the proportional Na

concentration in cell sap increased from 27% in

control to 50% in plants treated with 200 mM NaCl.

In contrast to cell sap, the two cultivars did not differ

in proportional Na concentration of apoplasmic fluid

and its change in response to salinity. Interestingly,

relative concentration (%) of Na in apoplasmic fluid

decreased under mild salinity in both cultivars

and further increased with increasing NaCl concen-

tration in the medium. Sodium concentration

in residual fraction (comprised mainly from cell

wall) was lower in salinity-treated IC than in 7233

(Table III).

Relative water content (RWC) of leaves was not

affected by salinity up to 50 mM, but it decreased in

7233 with NaCl treatments of 100 and 200 mM. In

IC, in contrast, RWC was reduced with higher

salinity only in tendency (Figure 2).

Effect of salinity on the antioxidant defence capacity

Salinity induced activity of APX in shoot and roots

at both low and higher NaCl concentrations with

Table I. Dry weight (mg plant�1) of shoot and root in six sugar beet (Beta vulgaris L.) cultivars grown for 21 days under four different NaCl

concentrations in nutrient solution. Reduction of DW was calculated between control and 200 mM NaCl treatment. Data of each cultivar

followed by the same letter are not significantly different (PB0.05).

Cultivar IC 7233 41-RT 276 Hybrid Rasoul

NaCl (mM) Shoot DW

Control 19109229a 17879144a 21139295a 14969236a 18309187a 15779255a

50 19679185a 11529170b 22969678a 12479200a 11689114b 13209251a

100 13209186b 10309105b 11009126b 10079170b 13879180b 11839161a

200 12319263b 6939155c 9039129b 737941b 758990c 600955b

LSD 0.05 12 14

Reduction (%) 36 61 57 51 58 62

Root DW

Control 518993a 386995a 453971a 314996a 354987a 314975a

50 487986a 249976ab 446991a 280957a 351973a 277961a

100 259993b 186933b 226933b 220969a 224976ab 306978a

200 278983b 161939b 146972b 170999a 148995b 113957b

Reduction (%) 46 58 68 46 58 64

Responses of sugar beet cultivars to salinity 251

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some differences between cultivars. Specific activity

of APX in roots was higher in IC than in 7233 in

plants treated with 200 mM NaCl. In contrast, APX

activity in roots of plants treated with 50 mM NaCl

was higher in 7233 than in IC. Such differential

response at different NaCl treatments was not

observed in shoots (Table IV).

Similarly, salinity induced activity of CAT in shoot

and root of both cultivars. However, mild salinity

caused reduction of CAT activity in roots of both

cultivars up to 45% and 53% in 7233 and IC,

respectively. In contrast to roots, salinity induced

CAT activity continuously in both levels of applied

NaCl in shoots (Table IV).

Peroxidase activity did not change significantly by

NaCl treatment in shoots, but increased at the

highest NaCl treatment in roots. In IC treated with

200 mM NaCl, POD activity of roots (relative to

corresponding control plants as well as absolutely)

was greater than in 7233 (Table IV).

Specific activity of SOD in shoots increased in

response to NaCl treatment, significantly in plants

treated with 200 mM NaCl. In roots of 7233, SOD

activity did not change significantly; in contrast,

0

400

800

1200

1600

Shoo

t D

W

7233

ab aa

b

c

0

400

800

1200

1600 IC

b

ab

a

bc

c

0

100

200

300

400

500

Roo

t D

W

a a

b

bc

c

0

100

200

300

400

500

aa

a

b b

0

100

200

300

400

500

Lea

f A

rea

aab

b

c

d

0

100

200

300

400

500b

aba

c

d

0

400

800

1200

NaCl (mM)

Chl

orop

hyll

ab

a

ab b

b

0

400

800

1200

0 25 50 100 2000 25 50 100 200NaCl (mM)

aa a ab

b

Figure 1. Shoot and root dry weight (mg plant�1), leaf area (cm2 plant�1), and chlorophyll concentration (mg g�1 FW) of two cultivars of

sugar beet (Beta vulgaris L. cvs 7233 and IC) treated for 14 days with different NaCl concentrations in the nutrient solution. Values are

mean9standard deviation (SD) from 4 replicates. Data of each measured parameters followed by the same letter are not significantly

different (P B 0.05).

252 R. Hajiboland & A. Joudmand

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SOD in roots of IC treated with 200 mM NaCl was

strongly increased (Table IV).

The effect of salinity on GR activity was mainly

insignificant with the exception of GR activity in the

roots of IC (Table IV).

An increase in H2O2 concentration was observed

in both shoots and roots and in both cultivars;

however, these changes were mainly significant only

at 200 mM NaCl treatment. Concentration of H2O2

in roots of plants treated with 200 mM NaCl in IC

was about 3-fold higher than that in 7233 (Table V).

Tissue concentration of superoxide radicals (O2.�)

increased with increasing NaCl treatment. At both

mild and high salinity treatments, concentration of

O2.� was orders of magnitude higher in 7233 than in

IC either in shoots or roots (Table V).

Concentration of MDA increased with increasing

salinity level, significantly in roots treated with 200

mM NaCl. Similarly with H2O2 and O2.�, the effect

of mild salinity level was mainly in tendency and

insignificant (Table V).

A significant accumulation of proline was ob-

served only at higher NaCl treatments. Proline

concentration was greater in roots than shoots in

both cultivars treated with 200 mM NaCl. A clear

difference was observed in proline accumulation of

shoots treated with 200 mM NaCl between two

cultivars; 7233 accumulated 2.3 times more proline

than did IC (Table V).

Leakage of K� from shoot and root tissues of IC

subjected to mild salinity (25 and 50 mM) was lower

than in control plants. In 7233, in contrast, NaCl

treatments higher than 25 mM caused leakage of K�

from both shoot and root tissues (Figure 3).

Effect of salinity on N metabolism

Protein concentration reduced with increasing NaCl

treatment level in tendency or significantly. Con-

siderable reduction of protein concentration up to

60% and 45% was observed in the presence of 200

mM NaCl in shoots of 7233 and roots of IC

cultivars, respectively. Salinity at high level (200

mM) decreased concentration of total free amino

acids in tendency in roots, but significantly in shoots

(Table VI).

Similarly, activity of NR did not change by mild

salinity but it responded to NaCl in both tested

cultivars at salt treatment of 200 mM. Reduction of

NR activity was similar in shoots of both cultivars

(55�58%) treated with 200 mM NaCl. Such reduc-

tion in roots was higher in IC (69%) than in 7233

(53%) (Table VI).

Discussion

In this work, salinity exerted a dual effect on plant

growth, depending on NaCl concentration in the

Table II. Concentration (mg g�1 DW) of Na, K and Ca in shoot and root of two cultivars of sugar beet (Beta vulgaris L. cvs 7233 and IC)

treated with different NaCl concentrations in the nutrient solution. Values are mean9standard deviation (SD) from 4 replicates. Data of

each cultivar followed by the same letter are not significantly different (PB0.05).

Shoot Root

Cultivar NaCl (mM) Na K Ca Na K Ca

7233 0 4.591.4c 10299a 0.4390.16b 3.193.1c 108911a 1.0890.05b

50 46.691.7b 10497a 0.5590.04ab 19.792.6b 109930a 1.6490.44a

200 70.992.3a 5099b 0.7090.02a 43.296.1a 60913b 2.0090.14a

IC 0 6.391.4c 140920a 0.3790.03b 7.391.9c 87920a 1.0090.06c

50 40.199.8b 82917b 0.5290.05b 21.693.7b 83914a 1.2890.13b

200 69.6918.7a 4997c 0.8290.15a 30.095.2a 64916a 1.8490.17a

Table III. Concentration of Na in leaf (mg g�1 FW) and leaf fractions including cell sap (mg g�1 FW) and apoplasmic fluid (mg g�1 FW)

in two cultivars of sugar beet (Beta vulgaris L. cvs 7233 and IC). Calculated values relative to total concentration are given in parentheses.

Values are mean9SD from 4 replicates. Data in each column followed by the same letter are not significantly different (PB0.05).

Cultivar

NaCl

(mM)

Total concentration

(mg g�1 FW)

Concentration in cell sap

(mg g�1 FW)

Concentration in apoplasmic

fluid (mg g�1 FW) Residual

Recovery

(%)

7233 0 0.8290.05 0.3790.09 (45) 0.1290.02 (15) 0.2790.02 (33) 83

50 8.8591.31 3.0090.32 (34) 0.1790.06 (2) 5.2291.33 (59) 92

200 26.9495.65 3.5090.45 (13) 2.9590.8 (11) 18.4493.51 (68) 90

IC 0 0.9490.05 0.2590.09 (27) 0.1190.01 (10) 0.5390.02 (56) 91

50 8.0291.09 3.6090.68 (45) 0.2890.03 (3) 3.5690.98 (44) 86

200 25.8593.28 12.9292.83 (50) 3.4990.9 (14) 8.9791.23 (35) 95

Responses of sugar beet cultivars to salinity 253

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medium. Moreover, a contrasting growth response

between two tested cultivars was observed under

both mild and severe salinity. Cultivars with higher

positive response to mild salinity were more tolerant

to higher salinity level and vice versa.

Effect of salinity on growth and its relationship with

concentration and distribution of ions

Compared with root and hypocotyl, shoot (leaf

weight and area) responded more strongly to mild

salinity. Surprisingly, the greater response to mild

salinity in IC was associated with significantly lower

K concentration in leaves. Although reduction of K

uptake in salinized plants is one of the main causes of

salt injury (Ashraf & Ahmad, 2000), it does not seem

to be a general mechanism particularly for includer

plants such as sugar beet. Lower K concentration in

leaves concomitant with growth improvement could

be considered the consequence of higher replace-

ment of K by Na in leaves of IC compared with 7233

under mild salinity. Sodium replaces potassium in its

contribution to the osmotic potential in the vacuoles

and consequently in the generation of turgor and cell

expansion (de Araujo et al., 2006). In addition, it

may surpass potassium in this respect since it

accumulates preferentially in the vacuoles. The

superiority of sodium was demonstrated by the

expansion of sugar beet leaf segments in vitro as

well as in intact sugar beet plants, where leaf area is

distinctly greater when a high proportion of potas-

sium is replaced by sodium (Marschner et al., 1981).

Induction of Ca deficiency in plants grown in

saline substrates and the role of Ca supplementation

in increasing salt tolerance of plants are well

documented (Porcelli et al., 1995; Cramer, 2004;

Shabala, 2005). The ameliorating effect of supple-

mental Ca in the medium is in accordance with its

function in membrane integrity and control of

selectivity in ion uptake and transport. High Na

concentration in the substrate inhibits uptake and

transport of Ca and may therefore induce calcium

deficiency in plants growing in substrates with low

Ca concentrations or high Na/Ca ratios (Munns,

2005). However, plant species differ considerably in

their sensitivity to Na-induced Ca deficiency (Cra-

mer, 2004). In this work, Ca was rather accumulated

in shoots and roots even under treatments causing

production of higher biomass, indicating that it is

not a concentration effect. This means that neither

mild nor severe salinity exerted an inhibitory effect

on Ca uptake and transport in sugar beet.

Salt treatment induced a reduction in the RWC of

leaves. This reduction was more pronounced in the

less tolerant cultivar, 7233, than in the more tolerant

one, IC. Reduction of RWC indicates a loss of turgor

that results in limited water availability for cell

expansion. Thus, the growth inhibition in 7233

could be related to reduction of RWC provoked by

the salt treatment.

Although leaf Na concentration did not differ in

the two tested cultivars, distribution of Na between

apoplasm and symplasm was differentially affected

by salinity depending on the cultivar. Proportional

Na in cell sap was higher under both mild and severe

salinity in cultivar IC than in 7233. In contrast, the

proportional Na in residual fraction comprised

mainly from cell wall was higher in 7233 than in

IC under both salinity levels. Allocation of more Na

to the cell sap (mainly vacuole) in IC may result in

facilitating control of water balance of leaf cells and

likely causes an improvement of cell expansion.

Production of broader leaves under mild salinity

and maintenance of high RWC under severe salinity

in IC are in agreement with this explanation. In

contrast to the presence of Na in the cell sap,

apoplasmic Na seems to have no role in differential

growth response of cultivars to salinity. It was

hypothesized that salt accumulation in the leaf

apoplasm is an important component of salt injury,

leading to dehydration and turgor loss and death of

leaf cells and tissues (Munns, 1988).

0

20

40

60

80

100

0 25 50 100 200NaCl Treatment (mM)

RW

C (

%)

7233 IC

Figure 2. Relative water content (%) of two cultivars of sugar beet (Beta vulgaris L. cvs 7233 and IC) treated with different NaCl

concentrations in the nutrient solution. Values are mean9SD from 4 replicates.

254 R. Hajiboland & A. Joudmand

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Functional significance of antioxidant capacity in

salinity response

Salinity was shown to alter the activity of antioxidant

system in roots (Lee et al., 2001; Khan et al., 2002).

In this work, response of the activity of antioxidant

enzymes and accumulation of related metabolites

and genotypic difference were more pronounced in

roots than in shoots. In sugar beet, shoot and root

likely have different contributions in determination

of whole-plant response to salinity. Shoot growth

and surface area of photosynthesizing organs is of

high importance for the supply of sugar for storage

roots. On the other hand, root growth could be of

great relevance because better growth causes higher

sink strength, phloem unloading, and consequently

higher sugar storage in roots (Van Bel, 1993). The

superiority of shoot over root or vice versa for

functioning as a determinant for sugar beet yield

under salinity has not been studied in detail and

needs more attention.

Activity of antioxidant enzymes was generally

induced by both salinity levels, in tendency or

significantly. Induction of antioxidant enzymes,

particularly those are effective in scavenging H2O2

(APX, CAT, and POD), resulted in obvious protec-

tion of plants and lowering of H2O2 accumulation in

tissues under mild salinity. However, under severe

salinity, though a higher activity of enzymes, more

H2O2 accumulated in leaves and roots.

Indigenous and salinity-induced accumulation of

O2.� was 2�3 orders of magnitude higher in 7233 in

both shoot and root, which was associated with

Table IV. Effect of NaCl salinity on the specific activity of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), superoxide

dismutase (SOD), and glutathione reductase (GR) in two cultivars of sugar beet (Beta vulgaris L. cvs 7233 and IC). Values are mean9SD

from 4 replicates. Data in each column within each plant part followed by the same letter are not significantly different (PB0.05).

APX (mmol H2O2 mg�1

protein min�1)

CAT (mmol H2O2 mg�1

protein min�1)

POD (mmol Guaiacol

mg�1 protein min�1)

SOD (Unit

mg�1 protein)

GR (nmol NADPH mg-1

protein min�1)

NaCl (mM) Shoot

0 8.992.9b 5679188b 10.492.2a 0.9290.23b 6.1891.28a

7233 50 16.294.7ab 10109297ab 10.893.2a 1.3290.45b 10.4493.03a

200 25.197.7a 14059471a 13.194.2a 3.4391.28a 9.0493.16a

0 7.792.4b 277939b 15.394.5a 1.8590.18b 9.7192.59a

IC 50 11.993.8ab 5869169ab 9.692.7a 2.9090.37ab 8.8691.11a

200 14.193.0a 8019251a 18.095.6a 4.0891.25a 8.4792.18a

Root

0 27.694.9b 4839151b 28.296.5b 1.4590.47a 3.9690.78a

7233 50 38.298.8ab 266952b 36.2911.1b 1.5390.29a 4.5391.18a

200 52.9910.8a 14669311a 76.1913.1a 1.6590.21a 5 .6491.75a

0 12.092.9b 276983b 41.0911.8b 1.0890.21b 2.7390.58b

IC 50 15.194.1b 127944b 52.5910.8b 1.3890.28b 9.5593.01b

200 85.8925.1a 27129809a 364.6978.1a 10.2991.29a 22.9795.36a

Table V. Effect of NaCl salinity on the concentration of H2O2, superoxide radicals, malondialdehyde (MDA), and proline in two cultivars of

sugar beet (Beta vulgaris L. cvs 7233 and IC). Values are mean9SD from 4 replicates. Data in each column within each plant part followed

by the same letter are not significantly different (PB0.05).

H2O2 (mg g�1 FW) O2.� (nmol g�1 FW) MDA (nmol g�1 FW) Proline (mmol g�1 FW)

NaCl (mM) Shoot

0 10.691.5b 158938b 21.995.9a 11.890.9b

7233 50 11.293.5b 312999ab 38.399.3a 11.392.7b

200 17.693.9a 5319166a 38.9911.3a 30.898.9a

0 12.792.6b 7997c 25.297.1a 10.092.7a

IC 50 14.994.1ab 134939b 31.1910.1a 10.292.1a

200 18.991.9a 223924a 44.1914.2a 13.193.9a

Root

0 2.190.9a 59917c 11.992.4b 12.893.2b

7233 50 3.090.9a 149932b 9.792.3b 16.194.6b

200 4.391.4a 353965a 33.6913.6a 45.0912.9a

0 2.590.8b 70912ab 12.992.8b 11.492.7b

IC 50 3.491.1b 123937a 10.390.8b 17.992.4b

200 13.193.3a 34985b 39.5910.5a 50.2911.9a

Responses of sugar beet cultivars to salinity 255

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lower activity of O2.�-scavenging enzyme, SOD. In

contrast, higher activity of SOD in IC caused

obviously lower accumulation of O2.�. More protec-

tion against O2.� radicals in IC as the result of higher

SOD activity could explain, at least partly, the

different responses of two tested cultivars particu-

larly to severe salinity. Several studies have demon-

strated that salt-tolerant species increase their

antioxidant enzyme activities and antioxidant con-

tents in response to salt stress, while salt-sensitive

species failed to do so (Meneguzzo et al., 1999;

Shalata et al., 2001).

Root concentration of proline under high-salinity

conditions was higher than for shoot. It could be

speculated that, in spite of shoot in which Na is an

osmoticum under salinity, proline plays that role in

0

10

20

30

40

50

K+

leak

age

(mg

g-1 D

W)

Control 25 mM 50 mM 100 mM 200 mM

7233

Control 25 mM 50 mM 100 mM 200 mM

7233

0

10

20

30

40

50

20 40 60 80 100 120 20 40 60 80 100 120Time (min)

K+

leak

age

(mg

g-1 D

W) IC

Time (min)

IC

Figure 3. Leakage of K� during 2 h measurement period from shoot (left) and root (right) tissues in two cultivars of sugar beet (Beta

vulgaris L. cvs 7233 and IC) treated with different NaCl concentrations. Values are mean9SD from 4 replicates.

Table VI. Effect of NaCl salinity on the concentration of protein (mg g�1 FW), total amino acids (TAA), and nitrate reductase activity

(NRA) in two cultivars of sugar beet (Beta vulgaris L. cvs 7233 and IC). Values are mean9SD from 4 replicates. Data in each column within

each plant part followed by the same letter are not significantly different (PB0.05).

Protein (mg g�1 FW) TAA (mmol g�1 FW) NRA (mmol NO2� g�1 FW)

NaCl (mM) Shoot

0 44.499.3a 203932a 15.092.6a

7233 50 38.6912.5ab 212971ab 18.094.4a

200 18.495.5b 109929b 6.392.3b

0 39.891.8a 204951a 20.096.8a

IC 50 33.993.0a 179942ab 14.794.8ab

200 29.299.4a 108931b 9.192.4b

Root

0 30.894.0a 3298a 9.492.5a

7233 50 30.195.9a 3495a 7.292.3ab

200 21.794.2a 2892a 4.491.4b

0 48.7914.9a 31918a 16.192.8a

IC 50 39.394.7ab 35912a 20.593.7a

200 26.791.8b 44917a 5.091.4b

256 R. Hajiboland & A. Joudmand

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roots. Proline concentration in shoots of 7233 was

2.3 times higher than in IC under the same NaCl

treatment (200 mM). Differences between 7233 and

IC in the contribution of Na and proline in osmotic

adjustment of leaves could be the cause/result of

different responses to NaCl. Since NaCl is a cheap

osmoticum with much lower energy cost for plants

compared with organic solutes (Marschner, 1995), a

preference of its accumulation over proline in

salinized plants may result a positive energy balance

and likely is one of the reasons for higher sugar

accumulation in roots observed under field condi-

tions (Hajiboland et al., unpublished data).

Interestingly, K� leakage from shoot and root

tissue of plants treated with low salinity was lower

than in control plants, which implies a beneficial role

for Na at low concentrations via a mechanism similar

to that shown by Se (Hartikainen et al., 2000). On

the other hand, the differential growth response of

two tested cultivars was well reflected in the amount

of K� leakage from shoot and root tissues. The

plasma membrane is the primary site of response to

salinity and may correlate with salt tolerance. There-

fore, change in plasma membrane permeability is a

good indicator for salt stress and tolerance and was

recommended as a reliable selection criterion for

developing salt-tolerant genotypes (Mansour & Sal-

ama, 2004). However, change in K� leakage did not

correlate with MDA concentration in our work. The

cause of this discrepancy is not known.

Protein synthesis in the leaves of plants growing in

saline substrates may decline in response to either a

water deficit or a specific ion excess (Thiyagarajah et

al., 1996). Replacement of K by Na may allow

osmotic adjustment in expanded leaves of salt-

tolerant species, but not the maintenance of protein

synthesis (Leidi & Saiz, 1997). Except in the case of

a few halophytes (Flowers & Dalmond, 1992; de

Araujo et al., 2006) Na cannot replace K in its

function in protein synthesis.

Moreover, lower total amino acids and protein

concentration could be accounted for by a reduction

of NR activity in salinized plants. Inhibition of NR

activity of salt-stressed plants was reported for maize

(Baki et al., 2000) and rice (Richharia et al., 2005).

Sugars are a source of reducing power for NR and

supply energy and carbon skeletons for the nitrogen-

assimilation process. Lower NR activity reduces

demand for photosynthates (Andreson & Peterson,

1988; Lam et al., 1996) and causes higher sucrose

accumulation (Werker et al., 1999).

According to our results, two major mechanisms

could be suggested for differences between responses

of two contrasting sugar beet cultivars to mild and

severe salinity. Allocation of Na to the cell sap and

consequently the amount of K replacement by Na in

this fraction, as well as protection against superoxide

radicals, were considerably different between the two

cultivars. However, mechanisms for growth stimula-

tion with and tolerance to mild and severe salinity

respectively, don’t seem to be related tightly to one

another. It seems likely that the first mechanism is

more important for the expression of growth im-

provement under mild salinity, and the second one

for an obviously greater tolerance to severe salinity.

Acknowledgements

Authors are greatful to K. Fotouhi, Center for Sugar

beet, West Azerbaijan Province, Iran for providing

sugar beet seeds.

References

Alia, P.S.P., & Mohanty, P. (1997). Involvement of proline in

protecting thylakoid membrane against free radicals induced

photodamage. Journal of Photochemistry & Photobiology B.

Biology, 38, 253�257.

Andreson, F.N., & Peterson, G.A. (1988). Effect of incrementing

nitrogen application on sucrose yield of sugar beet. Agronomy

Journal, 80, 709�712.

Apel, K., & Hirt, H. (2004). Reactive oxygen species: Metabo-

lism, oxidative stress, and signal transduction. Annual Review

of Plant Biology, 55, 373�399.

Ashraf, M., & Ahmad, S. (2000). Influence of sodium chloride on

ion accumulation, yield components and fiber characteristics

in salt-tolerant and salt-sensitive lines of cotton (Gossypium

hirsutum L.). Field Crops Research, 66, 115�127.

Baki, G.K. Abd-El., Siefritz, F., Man, H.-M., Weiner, H.,

Kaldenhoff, R., & Kaiser, W.M. (2000). Nitrate reductase

in Zea mays L. under salinity. Plant, Cell Environment, 23,

515�521.

Bartoz, G. (1997). Oxidative stress in plants. Acta Physiologia

Plantarum, 19, 47�64.

Bates, L.S., Waldren, S.P., & Teare, I.D. (1973). Rapid determi-

nation of free proline for water-stress studies. Plant and Soil,

39, 205�207.

Ben-Hayyim, G., Holland, D., & Eshdat, Y. (1999). Salt-induced

proteins related to oxidative stress: PHGPX and other

proteins of the Halliwell-Asada cycle. In M.F. Smallwood,

C.M. Calvert, & D.J. Bowles (Eds.), Plant Responses to

Environmental Stress (pp. 185�188). Bios Scientific Publish-

ers, Oxford, UK.

Bielski, B.H.J., Shlue, G.G., & Bajuk, S. (1980). Reduction of

nitro blue tetrazolium by CO2 and O2 radicals. Journal of

Physical Chemistry, 84, 830�833.

Boominathan, R., & Doran, P. (2002). Ni-induced oxidative stress

in roots of the Ni hyperaccumulator, Alyssum bertolonii. The

New Phytologist, 156, 205�215.

Bradford, M.M. (1976). A rapid and sensitive method for the

quantitation of microgram quantities of protein utilizing the

principle of protein-dye binding. Analytical Biochemistry, 72,

248�254.

Briat, J. F (2002). Metal ion-activated oxidative stress and its

control. In D. Inze, & M.V. Montgan (Eds.), Oxidative Stress

in Plants (pp. 171�190). Taylor & Francis, London, UK.

Chance, B., & Maehly, A.C. (1955). Assay of catalases and

peroxidases. Methods in Enzymology, 2, 764�775.

Cramer, G. (2004). Sodium-calcium interactions under salinity

stress. In A. Lauchli, & U. Luttge (Eds.), Salinity: Environ-

Responses of sugar beet cultivars to salinity 257

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go L

ibra

ry]

at 0

0:52

04

Oct

ober

201

3

ment-Plants-Molecules (pp. 205�227). Springer Verlag, Neth-

erlands.

Creissen, G.P., & Mullineaux, P.M. (2002). The molecular

biology of the ascorbate-glutathione cycle in higher plants.

In D. Inze, & M.V. Montgan (Eds.), Oxidative Stress in Plants

(pp. 247�270). Taylor & Francis, London, UK.

Cushman, J.C. (2001). Osmoregulation in Plants: Implications for

Agriculture. American Zoologist, 41, 758�769.

Dannel, F., Pfeffer, H., & Marschner, H. (1995). Isolation of

apoplasmic fluid from sunflower leaves and its use for studies

on influence of nitrogen supply on apoplasmic pH. Journal of

Plant Physiology, 146, 273�278.

de Araujo, S.A.M., Silveira, J.A.G., Almeida, T.D., Rocha,

I.M.A., Morais, D.L., & Viegas, R.A. (2006). Salinity

tolerance of halophyte Atriplex nummularia L. grown under

increasing NaCl levels. Revista Brasileira de Engenharia

Agrıcola e Ambiental, 10, 848�854.

De Vos, C.H.R., Schat, H., Vooijs, R., & Ernst, W.H.O. (1989).

Copper-Induced Damage to the Permeability Barrier in

Roots of Silene cucubalus. Journal of Plant Physiology, 135,

164�165.

Fadzilla, N.M., Finch, R.P., & Burdon, R.H. (1997). Salinity,

oxidative stress and antioxidant responses in shoot cultures

of rice. Journal of Experimental Botany, 48, 325�331.

Flowers, T.J., & Dalmond, D. (1992). Protein synthesis in

halophytes: The influence of potassium, sodium and magne-

sium in vitro. Plant and Soil, 146, 153�161.

Foyer, C.H., & Halliwell, B. (1976). The presence of glutathione

and glutathione reductase in chloroplasts: a proposed role in

ascorbic acid metabolism. Planta, 133, 21�25.

Ghoulam, C., Foursy, A., & Fares, K. (2002). Effects of salt stress

on growth, inorganic ions and proline accumulation in

relation to osmotic adjustment in five sugar beet cultivars.

Environmental & Experimental Botany, 47, 39�50.

Giannopolitis, C.N., & Ries, S.K. (1977). Superoxide dismutase

I. Occurrence in higher plants. Plant Physiology, 59, 309�314.

Gomez, J.M., Jimenez, A., Olmos, E., & Sevilla, F. (2004).

Location and effects of long-term NaCl stress on superoxide

dismutase and ascorbate peroxidase isoenzymes of pea

(Pisum sativum cv. Puget) chloroplasts. Journal of Experi-

mental Botany, 55, 119�130.

Gorham, J., & Jones, G. (2004). Genetics of Salinity Responses

and Plant Breeding. In A. Lauchli, & U. Luttge (Eds.),

Salinity: Environment-Plants-Molecules (pp. 451�489).

Springer Verlag, Netherlands.

Hartikainen, H., Xue, T., & Piironen, V. (2000). Selenium as an

anti-oxidant and pro-oxidant in ryegrass. Plant and Soil, 225,

193�200.

Heath, R.L., & Packer, L. (1968). Photoperoxidation in isolated

chloroplasts I. Kinetics and stoichiometry of fatty acid

peroxidation. Archives in Biochemistry & Biophysics, 125,

189�198.

Hoagland, D.R., & Arnon, D.I. (1950). The water culture method

for growing plants without soil. Circular 347 of the Califor-

nia Agricultural Experimental Station Circular, Berkeley,

CA, USA.

Hwang, M., & Ederer, G.M. (1975). Rapid hippurate hydrolysis

method for presumptive identification of group B strepto-

cocci. Journal of Clinical Microbiology, 1, 114�115.

Jaiswal, P.C. (2004). Soil, Plant and Water Analysis. Kalyani

Publishers, New Delhi, India.

Jaworski, E.G. (1971). Nitrate reductase assay in intact plant

tissue. Biochemical & Biophysical Research Communications,

43, 1274�1279.

Jimenez, A., Hernandez, J.A., del Rıo, L.A., & Sevilla, F. (1997).

Evidence for the presence of the ascorbate-glutathione cycle

in mitochondria and peroxisomes of pea leaves. Plant

Physiology, 114, 275�284.

Khan, M.H., Singha, K.L.B., & Panda, S.K. (2002). Changes in

antioxidant levels in Oryza sativa L. roots subjected to NaCl-

salinity stress. Acta Physiologiae Plantarum, 24, 145�148.

Lam, H.-M., Coschigani, K.T., Oliveira, I.C., Melo-Oliveira, R.,

& Coruzzi, G.M. (1996). The molecular genetics of nitrogen

assimilation into amino acids in higher plants. Annual Review

of Plant Physiology and Plant Molecular Biology, 47, 569�593.

Lee, D.H., Kim, Y.S., & Lee, C.B. (2001). The inductive

responses of the antioxidant enzymes by salt stress in the

rice (Oryza sativa L.). Journal of Plant Physiology, 158, 737�745.

Leidi, E.O., & Saiz, J.F. (1997). Is salinity tolerance related to Na

accumulation in Upland cotton (Gossypium hirsutum) seed-

lings. Plant and Soil, 190, 67�75.

Luck, H. (1962). In H.U. Bergmeyer (Ed.), Methods of Enzymatic

Analysis (pp. 885�894). VCH, Weinheim, Germany.

Mansour, M.M., & Salama, K.H.A. (2004). Cellular basis of

salinity tolerance in plants. Environmental & Experimental

Botany, 52, 113�122.

Marschner, H. (1995). Mineral Nutrition of Higher Plants (2nd

edn). Academic Press, London, UK.

Marschner, H., Kylin, A., & Kuiper, P.J.C. (1981). Genotypic

differences in the response of sugar beet plants to replace-

ment of potassium by sodium. Physiologia Plantarum, 51,

239�244.

Meloni, D.A., Oliva, M.A., Martinez, C.A., & Cambraia, J.

(2003). Photosynthesis and activity of superoxide dismutase,

peroxidase and glutathione reductase in cotton under salt

stress. Environmental & Experimental Botany, 49, 69�76.

Meneguzzo, S., Navari-Izzo, F., & Izzo, R. (1999). Antioxidative

responses of shoots and roots of wheat to increasing NaCl

concentrations. Journal of Plant Physiology, 155, 274�280.

Moran, R. (1982). Formulae for determination of chlorophyllous

pigments extracted with N,N-dimethylformamide. Plant

Physiology, 69, 1376�1381.

Munns, R. (1988). Effect of high external NaCl concentrations on

ion transport within the shoot of Lupinus albus. I. Ions in

xylem sap. Plant, Cell & Environment, 11, 283�289.

Munns, R. (2005). Genes and salt tolerance: bringing them

together. The New Phytologist, 167, 645�663.

Patterson, B.D., MacRae, E.A., & Ferguson, I.B. (1984). Estima-

tion of hydrogen peroxide in plant extracts using titanium

(IV). Analytical Biochemistry, 139, 487�492.

Pitman, M., & Lauchli, A. (2004). Global impact of salinity and

agricultural ecosystems. In A. Lauchli, & U. Luttge (Eds.),

Salinity: Environment-Plants-Molecules (pp. 3�20). Springer

Verlag, Netherlands.

Porcelli, C.A., Gutierrez Boem, F.H., & &Lavado, R.S. (1995).

The K/Na and Ca/Na ratios and rapeseed yield, under soil

salinity or sodicity. Plant and Soil, 175, 251�255.

Rhodes, D., Nadolska-Orczyk, A., & Rich, P.J. (2004). Salinity,

osmolytes and compatible solutes. In A. Lauchli, & U. Luttge

(Eds.), Salinity: Environment -Plants-Molecules (pp. 181�204).

Netherlands, Springer Verlag.

Richharia, A., Shah, K., & Dubey, R.S. (2005). Nitrate reductase

from rice seedlings: Partial purification, characterization and

the effects of in situ and in vitro NaCl salinity. Journal of Plant

Physiology, 162, 854�864.

Santos, C.L.V., Campos, A., Azevedo, H., & Caldeira, G. (2001).

In situ and in vitro senescence induced by KCl stress:

258 R. Hajiboland & A. Joudmand

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go L

ibra

ry]

at 0

0:52

04

Oct

ober

201

3

nutritional imbalance, lipid peroxidation and antioxidant

metabolism. Journal of Experimental Botany, 52, 351�360.

Shabala, S., Shabala, L., Van Volkenburgh, E., & Newman, I.

(2005). Effect of divalent cations on ion fluxes and leaf

photochemistry in salinized barley leaves. Journal of Experi-

mental Botany, 56, 1369�1378.

Shalata, A., Mittova, V., Volokita, M., Guy, M., & Tal, M. (2001).

Response of the cultivated tomato and its wild salt-tolerant

relative Lycopersicon pennellii to salt-dependent oxidative

stress: the root antioxidative system. Physiologia Plantarum,

112, 487�494.

Thiyagarajah, M., Fry, S.C., & Yeo, A.R. (1996). In vitro salt

tolerance of cell wall enzymes from halophytes and glyco-

phytes. Journal of Experimental Botany, 47, 1717�1724.

Van Bel, A.J.E. (1993). Strategies of phloem loading. Annual

Review of Plant Physiology & Plant Molecular Biology, 44,

253�281.

Werker, A.R., Jaggard, K.W., & Allison, M.F. (1999). Modeling

partitioning between structure and storage in sugar beet:

Effects of drought and soil nitrogen. Plant and Soil, 207,

97�106.

Responses of sugar beet cultivars to salinity 259

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go L

ibra

ry]

at 0

0:52

04

Oct

ober

201

3