Salt Stress Induces Various Biochemical Biology Essay

Introduction

Salt stress induces various biochemical and

physiological responses in plants and affects almost

all plant processes (Nemoto and Sasakuma,

2002). Salinity can cause hyperionic and hyperosmotic

effects in plants leading to membrane disorganization,

increase in reactive oxygen species

(ROS) levels, and metabolic toxicity (Jaleel et al.,

2007). High-salt stress disrupts the homeostasis in

water potential and ion distribution at both the

cellular and the whole plant levels (Errabii et al.,

2007). Excess of Na+ and Cl- ions may lead to

conformational changes in the protein structure,

while osmotic stress leads to turgor loss and cell

volume change (Errabii et al., 2007). However,

the precise mechanisms underlying these effects

are not fully understood because the resistance

to salt stress is a multigenic trait (Errabii et al.,

2007). To achieve salt tolerance, plant cells evolve

several biochemical and physiological pathways.

These processes are thought to operate additively

to ensure plants’ and cells’ survival, and

they include the exclusion of Na+ ions and their

compartmentation into vacuoles as well as accumulation

of compatible solutes such as proline,

glycinebetaine, and polyols (Errabii et al., 2007).

In order to survive under stress conditions, plants

are equipped with oxygen radical-detoxifying

enzymes such as superoxide dismutase (SOD),

ascorbate peroxidase (APX), catalase (CAT), and

glutathione reductase (GR). Oxidative stress is

the result of ROS, such as superoxide, H2O2, and

hydroxyl radicals, and causes rapid cell damage

by triggering off a chain reaction. ROS scavenging

is one among the common defense responses

against abiotic stresses. Changes in antioxidants

and protective molecules refl ect the impact of environmental

stresses on plant metabolism (Jaleel

et al., 2007). Salt-tolerant plants, besides being

Effective Salt Criteria in Callus-Cultured Tomato Genotypes

Mahmut Dogana, Rukiye Týpýrdamazb, and Yavuz Demirc,*

a Harran Üniversitesi, Fen Edebiyat Fakültesi, Biyoloji Bölümü, Osmanbey Kampüsü,

Sanlýurfa, Turkey

b Hacettepe Üniversitesi, Fen Fakültesi, Biyoloji Bölümü, Beytepe Kampüsü, Ankara,

Turkey

c Atatürk Üniversitesi, K. K. Eðitim Fakültesi, Biyoloji Bölümü, 25240-Erzurum, Turkey.

Fax: (+90) 44 22 36 09 55. E-mail: ydemir_409@yahoo.com

* Author for correspondence and reprint requests

Z. Naturforsch. 65 c, 613 – 618 (2010); received January 27/May 7, 2010

Na+, Cl–, K+, Ca2+, and proline contents, the rate of lipid peroxidation level in terms of

malondialdehyde (MDA) and chlorophyll content, and the changes in the activity of antioxidant

enzymes, such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC

1.11.1.6), ascorbate peroxidase (APX: EC 1.11.1.11), and glutathione reductase (GR: EC

1.6.4.2), in tissues of fi ve tomato cultivars in salt tolerance were investigated in a callus

culture. The selection of effective parameters used in these tomato genotypes and to fi nd

out the use of in vitro tests in place of in vivo salt tolerance tests were investigated. As a

material, fi ve different tomato genotypes during a 10-day time period were used, and 150 mM

NaCl was applied at callus plant tissue. The exposure to NaCl induced a signifi cant increase

in MDA content in both salt-resistant and salt-sensitive cultivars. But the MDA content

was higher in salt-sensitive cultivars. The chlorophyll content was more decreased in saltsensitive

than in salt-resistant ones. The proline amount was more increased in salt-sensitive

than in salt-resistant ones. It has been reported that salt-tolerant plants, besides being able

to regulate the ion and water movements, also exhibit a strong antioxidative enzyme system

for effective removal of ROS. The degree of damage depends on the balance between the

formation of ROS and its removal by the antioxidative scavenging system that protects

against them. Exclusion or inclusion of Na+, Cl–, K+, and Ca2+ , antioxidant enzymes and

MDA concentration play a key protective role against stress, and this feature at the callus

plant tissue used as an identifi er for tolerance to salt proved to be an effective criterion.

Key words: Antioxidant Enzyme, Salinity, Tomato

614 M. Dogan et al. · Salt-Tolerant Tomato Genotypes

able to regulate the ion and water movements,

should also have a better antioxidative system for

effective removal of ROS and higher activities of

SOD, APX and glutathione transferase (GST) enzymes

of chloroplasts which probably play a key

role in defense against oxidative damage (Wang

et al., 2008).

The mechanisms of salt tolerance, not yet clear,

can be, to some extent, explained by stress adaptation

effectors that mediate the ion homeostasis,

osmolytic biosynthesis, toxic radical scavenging,

water transport, and long-distance response coordination

(Jaleel et al., 2007). Undoubtedly, plant

breeders have made a signifi cant achievement

in the past few years, which improves the salinity

tolerance in a number of potential crops using

artifi cial selection and conventional breeding

approaches. However, most of the selection procedures

have been based on differences in agronomic

characters (Ashraf and Harris, 2004). Agronomic

characters represent the combined genetic

and environmental effects on plant growth and

include integration of the physiological mechanisms

conferring salinity tolerance. Typical agronomic

selection parameters for salinity tolerance

are yield, survival, plant height, leaf area, leaf injury,

relative growth rate, and relative growth reduction

(Ashraf and Harris, 2004). Many scientists

have suggested that selection is more convenient

and practicable if the plant species possesses distinctive

indicators of salt tolerance at the whole

plant, tissue or cellular level (Ashraf and Harris,

2004).

In recent years, tissue culture has gained importance

in the development of plants against

various abiotic stresses as well as in elucidating

mechanisms operating at the cellular level by

which plants survive under various abiotic stresses

including salinity (Jain et al., 2001). Plant tissue

culture allows to control the stress homogeneity

and to characterize the cell behaviour under

stress conditions, independently of the regulatory

systems that take place at the whole plant level

(Lutts et al., 2004).

The objective of the present investigation was

to study the effect of salinity stress on Na+, Cl–,

K+, Ca2+, and proline contents, the rate of lipid

peroxidation level in terms of malondialdehyde

(MDA) and chlorophyll content, and the plant

antioxidant systems (SOD, CAT, APX, and GR)

in relatively salt-sensitive and -tolerant tomato

cultivars in order to evaluate the relative signifi -

cance of these parameters in imparting tolerance

to NaCl oxidative stress.

Material and Methods

Growth conditions

Seeds of fi ve tomato genotypes, four of which

belonged to the local Lycopersicum esculentum

species (TR-47815 L. esculentum Turkey; TR-

47882 L. esculentum Turkey; TR-55711 L. esculentum

Turkey; TR-68516 L. esculentum Turkey) and

the other one belonged to the L. peruvianum wild

species (PI-899-01 L. peruvianum), were used.

The seeds were cleaned from bacteria and fungi

by applying the superfi cial sterilization method of

Ellis et al. (1988). Then six seeds of each species

were put in magenta pots containing MS basal

nourishment medium.

Explants collected from hypocotyls by cutting

from the root neck of the seedlings after

the formation of the fi rst three leaves in a climate

chamber were planted horizontally in Petri

dishes containing MS medium supplemented with

1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D)

and 0.1 mg/l kinetin. Petri dishes were placed in

a climate chamber at (25 ± 2) °C under permanent

dark conditions. When the root length of the

seedlings reached 1 cm, magentas were held in a

16 h light/8 h dark photoperiod. Calli, formed approx.

within 4 weeks, were placed in the subculture

by being separated from the main explants.

After 4 weeks, they were placed into nourishment

medium containing 150 mM NaCl. Calluses, completing

within 4 weeks in the subculture, were

transferred into 15 Petri dishes as control. In

each Petri dish, 8 callus tissues, each of 1 g, were

placed. They were left to grow for 10 d in a medium

containing 150 mM NaCl. At the end of the

10th day, the harvested leaves were kept in a deep

freezer at – 80 °C and homogenized in liquid nitrogen

to do the enzyme analyses. Extracts were

prepared from the calli of the control and salt-applied

plants, and enzyme activities in the obtained

supernatant were determined (Cakmak, 1994).

A randomized parcels experimental design

was carried out with three replications in factorial

order. Time, one of these three factors, has a

replicated measurement quality. The factors were

compared by the repeated measurement variance

analysis from the view point of the properties

concerned. The differences between the levels of

the factors were examined by the least important

M. Dogan et al. · Salt-Tolerant Tomato Genotypes 615

difference (L.I.D.) multiple comparison method.

The calculations were made using MINITAB 13.0

statistical packet program. Statistica V.6.0 packet

program was used for multiple comparisons of

the properties.

Due to the homogeneous structure of the callus

tissue and its providing convenience to study in

a small area in a short time with a great number

of plant materials, the impression of likelihood of

preference of callus cultures in stress studies was

got. Relying on the ideas above, the reactions of

local genotypes to salt, TR-68516 and TR-55711

of L. esculentum, were determined to be similar

to that of the L. peruvianum wild species, and

these genotypes were classifi ed as salt-tolerant.

TR-47815 and TR-47882 belonging to L. esculentum

were decided as salt-sensitive and were determined

as the most different genotypes.

Determination of ion content

For ion measurements, calli were fi rst rinsed for

5 min with cool distilled water in order to remove

free ions from the apoplasm without substantial

elimination of cytosolic solutes. Calli were ovendried

at 80 °C for 48 h and then were ground. The

dry matter obtained was used for mineral analysis.

The major cations were extracted after digestion

of dry matter with HNO3. The extract was fi ltered

prior to analysis. Na+ and K+ contents were

determined using a fl ame spectrophotometer. The

Ca2+ concentration was quantifi ed by an atomic

absorption spectrophotometer (Shimadzu AA-

6200, Kyoto, Japan) (Guerrier and Patolia, 1989).

For Cl- content estimation, ions were extracted

with hot distilled water (80 °C during 2 h). Chloride

was determined as described by Taleisnik et

al. (1997).

Chlorophyll content

The total chlorophyll content was analysed according

to Luna et al. (2000).

Lipid peroxidation

For measurement of the lipid peroxidation in

leaves, the MDA content (red pigment) was determined

according to Lutts et al. (1996).

Proline content

The amount of proline was determined according

to the method of Bates et al. (1973).

Enzyme assay

To determine the enzyme activities, 0.5 g of

leaf tissues from control and treated plants were

ground in liquid nitrogen and homogenized in

3 ml of buffer containing 50 mM KH2PO4 buffer

(pH 7.0), 0.1 mM EDTA, and 1% PVPP (w/v).

The homogenates were centrifuged at 15,000 ×

g for 15 min at 4 °C, and the resulting supernatants

were freshly used for determination of SOD,

CAT, GR, and APX activities.

The SOD (EC 1.15.1.1) activity was measured

by the modifi ed method of Beyer and Fridovich

(1987). 3 ml of the reaction mixture contained

50 mM phosphate buffer (pH 7.8), 13 mM methionine,

60 µM nitroblue tetrazolium (NBT),

0.1 mM EDTA, and 100 µl enzyme extract. The

reaction was started by adding 60 µM ribofl avine

and placing the tubes under two 20-W cool white

fl uorescent lamps for 30 min. A complete reaction

mixture without enzyme served as control.

The reaction was stopped by switching off the

light and putting the tubes into the dark. A nonirradiated

completed reaction mixture served as

a blank. The absorbance was recorded at 560 nm.

One unit of SOD enzyme was defi ned as the

amount that produces 50% inhibition of NBT reduction

under the assay conditions and expressed

as U SOD activity mg–1 protein.

For determination of the CAT (EC 1.11.1.6)

activity, the reaction mixture contained 50 mM

KH2PO4 (pH 7), 13 mM H2O2, and 30 µl enzyme

extract. The decrease in absorbance of H2O2 was

recorded at 240 nm for 3 min using a spectrophotometer

(Shimadzu UV-VIS-1208). One unit

of activity was defi ned as the amount of enzyme

catalyzing the decomposition of 1 µmol H2O2 per

min, calculated from the extinction coeffi cient

(0.036 cm2/µmol) for H2O2 at 240 nm (Öztürk and

Demir, 2003).

The GR (EC 1.6.4.2) activity was measured

according to Foyer and Halliwell (1976). The assay

medium contained 0.025 mm sodium phosphate

buffer (pH 7.8), 0.5 mM GSSG, 0.12 mM

NADPHNa4, and 100 µl enzyme extract in a fi nal

assay volume of 1 ml. NADPH oxidation was determined

at 340 nm. The activity was calculated

using the extinction coeffi cient . = 6.2 mM-1 cm-1

for GSSG. One unit of GR activity was defi ned as

1 mmol/ml GSSG reduced per min.

The activity of APX (EC 1.11.1.11) was measured

according to the method of Karabal et al.

616 M. Dogan et al. · Salt-Tolerant Tomato Genotypes

(2003). The reaction mixture consisted of 50 mM

phosphate buffer (pH 6), 1.47 mM H2O2, 0.5 mM

ascorbic acid, and 50 µl enzyme extract. The reaction

was started by the addition of H2O2, and the

oxidation of ascorbate was measured for 3 min at

290 nm. The enzyme activity was calculated from

the initial rate of the reaction using the extinction

coeffi cient 2.8 mM–1 cm–1 at 290 nm for ascorbate.

Statistical analysis

Tables indicate mean values ± SE. Differences

between the values for control and treated leaves

were analysed by one-way ANOVA, taking P <

0.001 as signifi cance level, according to LSD multiple

range tests.

Results and Discussion

In the absence of stress, in all tissues, the Na+

level differed signifi cantly (P < 0.001) among the

cultivars and was lower in salt-resistant than in

salt-sensitive cultivars, while no signifi cant difference

was recorded among cultivars in reffering

to the Cl- level. The exposure to NaCl induced a

signifi cant increase in Na+ and Cl- levels in both

salt-resistant and salt-sensitive cultivars (Table I).

Table I. Na+ (µg/mg dry weight) and Cl– (µg/mg dry weight) contents in roots, stems, and leaves of the L. esculentum

species in the presence of 150 mM NaCl. Results are expressed as means ± SE (n = 3).

Genotype Roots Stems Leaves

Control Salt Control Salt Control Salt

TR-68516 Na+ 3.6 ± 0.2 4.8 ± 0.3 3.5 ± 0.2 4.5 ± 0.3 3.3 ± 2 4.2 ± 0.3

Cl– 3 ± 0.1 4 ± 0.1 9 ± 0.1 12 ± 0.1 2 ± 0.1 3 ± 0.1

TR-55711 Na+ 3.6 ± 0.2 4.7 ± 0.2 3.4 ± 0.1 4.4 ± 0.2 3.2 ± 1 4 ± 0.2

Cl– 3 ± 0.1 5 ± 0.1 3 ± 0.1 5 ± 0.1 2 ± 0.1 3 ± 0.1

PI-899-01 Na+ 3.8 ± 0.4 5 ± 0.6 3.6 ± 0.3 4.5 ± 0.6 3.3 ± 3 4.2 ± 0.4

Cl– 2 ± 0.1 3 ± 0.1 20 ± 0.1 29 ± 0.1 12 ± 0.1 18 ± 0.1

TR-47815 Na+ 9.8 ± 0.6 15.8 ± 0.8 9.2 ± 0.5 14.8 ± 0.8 8.7 ± 5 13.9 ± 0.7

Cl– 2 ± 0.1 5 ± 0.1 2 ± 0.1 4 ± 0.1 2 ± 0.1 4 ± 0.1

TR-47882 Na+ 9.7 ± 0.1 15.5 ± 0.2 9.1 ± 0.1 14.6 ± 0.2 8.6 ± 1 13.7 ± 0.2

Cl– 3 ± 0.1 8 ± 0.1 3 ± 0.1 7 ± 0.1 3 ± 0.1 7 ± 0.1

Na+ LSD (genotype . NaCl

treatment) (P < 0.001): 2.2

LSD (genotype. NaCl

treatment) (P < 0.001): 2.3

LSD (genotype. NaCl

treatment) (P < 0.01): 2.64

Cl– LSD (genotype . NaCl

treatment) (P < 0.001): 2.5

LSD (genotype . NaCl

treatment) (P < 0.001): 2.3

LSD (genotype . NaCl

treatment) (P < 0.01): 1.8

Table II. K+ (µg/mg dry weight) and Ca2+ (µg/mg dry weight) contents in roots, stems, and leaves of the L. esculentum

species in the presence of 150 mM NaCl. Results are expressed as means ± SE (n = 3).

Genotype Roots Stems Leaves

Control Salt Control Salt Control Salt

TR-68516 K+ 3.7 ± 0.1 5.9 ± 0.1 3.4 ± 0.1 5.5 ± 0.1 3.2 ± 0.1 5.2 ± 0.1

Ca2+ 38 ± 0.7 66 ± 0.8 31 ± 0.9 57 ± 0.8 20 ± 0.8 33 ± 0.4

TR-55711 K+ 4.3 ± 0.1 6.9 ± 0.1 4.5 ± 0.1 6.5 ± 0.1 3.7 ± 0.1 6.1 ± 0.1

Ca2+ 34 ± 0.8 59 ± 0.9 30 ± 0.6 55 ± 0.6 24 ± 0.9 39 ± 1.1

PI-899-01 K+ 4.2 ± 0.1 6.7 ± 0.1 4 ± 0.1 6.2 ± 0.1 3.8 ± 0.1 5.8 ± 0.1

Ca2+ 37 ± 1.6 65 ± 0.7 35 ± 0.6 65 ± 0.6 23 ± 0.9 38 ± 1.2

TR-47815 K+ 3 ± 0.1 4.8 ± 0.1 2.8 ± 0.1 4.5 ± 0.1 2.6 ± 0.1 4.2 ± 0.1

Ca2+ 35 ± 0.8 38 ± 2.4 29 ± 0.6 32 ± 0.6 17 ± 0.6 21 ± 1.2

TR-47882 K+ 2 ± 0.1 3.3 ± 0.1 1.9 ± 0.1 3.1 ± 0.1 1.8 ± 0.1 2.9 ± 0.1

Ca2+ 56 ± 2.1 60 ± 1.9 49 ± 1.5 55 ± 0.5 41 ± 0.5 48 ± 0.6

K+ LSD (genotype . NaCl

treatment) (P < 0.001): 0.6

LSD (genotype . NaCl

treatment) (P < 0.001): 0.5

LSD (genotype . NaCl

treatment) (P < 0.01): 0.5

Ca2+ LSD (genotype . NaCl

treatment): 1.7

P < 1.2

LSD (genotype . NaCl

treatment): 0.95

P < 0.6

LSD (genotype . NaCl

treatment): 0.8

P < 0.7

M. Dogan et al. · Salt-Tolerant Tomato Genotypes 617

But the Na+ and Cl- levels were higher in saltsensitive

cultivars.

In the absence of stress, in all tissues, the K+

level differed signifi cantly (P < 0.001) among

the cultivars and was lower in salt-sensitive than

in salt-resistant cultivars. The exposure to NaCl

induced a signifi cant increase in the K+ levels

in both salt-resistant and salt-sensitive cultivars

(Table II). In this study, only the salt-tolerant cultivars

maintained higher K+ contents and lower

Na+ contents in all tissues of the plant (Tables I

and II).

In the absence of stress, in all tissues, the Ca2+

level differed signifi cantly (P < 0.001) among the

cultivars and was lower in salt-sensitive than in

salt-resistant cultivars. The exposure to NaCl induced

a signifi cant increase in the Ca2+ level in

both salt-resistant and salt-sensitive cultivars

(Table II). But the Ca2+ level was higher in saltresistant

cultivars.

The lipid peroxidation levels in leaves of the

fi ve tomato cultivars, measured as the content

of MDA, are given in Table III. In the absence

of stress, the MDA level differed signifi cantly

(P < 0.01) among the cultivars and was higher in

salt-resistant than in salt-sensitive cultivars. The

exposure to NaCl induced a signifi cant increase

in the MDA level in both salt-resistant and saltsensitive

cultivars. But the MDA level was higher

in salt-sensitive cultivars. The better NaCl stress

tolerance in salt-tolerant cultivars as compared to

salt-sensitive cultivars observed during the present

investigation may be due to the restriction of

damage to cellular membranes with lower MDA

and H2O2 content.

In the absence of stress, the chlorophyll level in

leaf tissue differed signifi cantly (P < 0.01) among

the cultivars and was higher in salt-resistant than

in salt-sensitive cultivars (Table III). The exposure

to NaCl induced a signifi cant decrease in the

chlorophyll level in both salt-resistant and saltsensitive

cultivars. But the chlorophyll level was

more decreased in salt-sensitive cultivars than in

salt-resistant cultivars.

In the absence of stress, the proline level differed

signifi cantly (P < 0.01) among the cultivars

and was higher in salt-resistant than in salt-sensitive

cultivars (Table III). The exposure to NaCl

induced a signifi cant increase in the proline level

in both salt-resistant and salt-sensitive cultivars.

Table III. Malondialdehyde (MDA) (µmol/g fresh weight), chlorophyll (µg/g fresh weight), and proline (µmol/g

fresh weight) contents in leaves of the L. esculentum species in the presence of 150 mM NaCl. Results are expressed

as means ± SE (n = 3).

Genotype MDA Chlorophyll Proline

Control Salt Control Salt Control Salt

TR-68516 32 ± 0.2 35 ± 0.3 326 ± 0.2 298 ± 0.2 30 ± 0.2 33 ± 0.3

TR-55711 26 ± 0.1 28 ± 0.1 328 ± 0.1 308 ± 0.4 25 ± 0.1 27 ± 0.1

PI-899-01 32 ± 0.2 33 ± 0.2 331 ± 0.2 312 ± 0.1 31 ± 0.2 34 ± 0.2

TR-47815 24 ± 0.2 44 ± 0.3 233 ± 1.3 123 ± 0.1 25 ± 0.2 39 ± 0.3

TR-47882 25 ± 0.1 45 ± 0.1 232 ± 0.1 140 ± 0.2 26 ± 0.1 43 ± 0.1

LSD (genotype . NaCl

treatment) (P < 0.01): 1.55

LSD (genotype . NaCl

treatment) (P < 0.01): 1.33

LSD (genotype . NaCl

treatment) (P < 0.01): 1.55

Table IV. The differences between the superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR),

and ascorbate peroxidase (APX) enzyme activities [µmol/(min mg fresh weight)] of the callus tissues of the different

tomato genotypes according to the control. Values are given as the means of three repetitions ± standard

deviation.

Genotype SOD CAT GR APX

Control Salt Control Salt Control Salt Control Salt

TR-68516 144 ± 4 390 ± 12 43 ± 0.1 155 ± 0.1 125 ± 0.0 259 ± 0.1 111 ± 0.2 249 ± 0.1

TR-55711 184 ± 4 356 ± 23 52 ± 0.1 81 ± 0.1 136 ± 0.1 259 ± 0.1 124 ± 0.1 256 ± 0.1

PI-899-01 118 ± 6 357 ± 19 33 ± 0.1 311 ± 0.2 138 ± 0.2 288 ± 0.2 127 ± 0.1 273 ± 0.6

TR-47815 114 ± 8 138 ± 56 34 ± 0.1 39 ± 0.2 122 ± 0.1 215 ± 0.2 122 ± 0.0 198 ± 0.0

TR-47882 142 ± 4 167 ± 42 49 ± 0.1 52 ± 0.1 143 ± 0.2 216 ± 0.1 129 ± 0.3 154 ± 0.01

LSD (genotype . NaCl treatment): 7.41 (SOD), 2.6 (CAT), 3.2 (GR), 2.6 (APX)

618 M. Dogan et al. · Salt-Tolerant Tomato Genotypes

But the proline level was more increased in saltsensitive

cultivars than in salt-resistant ones.

In the absence of stress, the SOD, CAT, GR,

and APX activities differed signifi cantly among

the cultivars (Table IV). The exposure to NaCl

induced a signifi cant increase in the enzyme activities

in both salt-resistant and salt-sensitive cultivars.

But the enzyme activities were signifi cantly

higher in salt-resistant cultivars.

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