Plant Science 135 (1998) 1 – 9

Antioxidant responses of rice seedlings to salinity stress

Maribel L. Dionisio-Sese *, Satoshi Tobita 1

Okinawa Subtropical Station, Japan International Research Center for Agricultural Sciences (JIRCAS), Ishigaki,
Okinawa 907, Japan

Received 14 October 1997; received in revised form 31 January 1998; accepted 31 January 1998

Abstract

The possible involvement of activated oxygen species in the mechanism of damage by NaCl stress was studied in
leaves of four varieties of rice (Oryza sati6a L.) exhibiting different sensitivities to NaCl. The 3-week-old rice seedlings
were subjected to 0, 6 and 12 dS m − 1 salinity levels for 1-week after which differences in antioxidant capacities and
possible correlation, growth rate and Na + uptake of the leaves were analyzed. High salinity treatment caused a
decrease in growth rate in all the varieties tested except Pokkali. The salt-sensitive varieties, Hitomebore and IR28,
exhibited a decrease in superoxide dismutase activity and an increase in peroxidase activity under high salinization.
These varieties also exhibited increase in lipid peroxidation and electrolyte leakage as well as higher Na +
accumulation in the leaves under salt stress. The salt-tolerant variety Pokkali however, showed only slight increase
and decrease in superoxide dismutase and peroxidase activity, respectively, and virtually unchanged lipid peroxida-
tion, electrolyte leakage and Na + accumulation upon salinization. On the other hand, the putative salt-tolerant
Bankat variety, which showed a slight stimulation in growth rate similar to Pokkali at moderate salinity level,
exhibited Na + accumulation and symptoms of oxidative damage during salt stress similar to the salt-sensitive
varieties rather than the salt-tolerant one. These results indicate that free radical-mediated damage of membrane may
play an important role in the cellular toxicity of NaCl in rice seedlings and that salt-tolerant varieties exhibit
protection mechanism against increased radical production by maintaining the specific activity of antioxidant
enzymes. © 1998 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Lipid peroxidation; Oryza sati6a L.; Oxidative stress; Peroxidase; Salt stress; Superoxide dismutase

1. Introduction

* Corresponding author. Present address: Institute of Bio- Salinity in soil or water is of increasing impor-
logical Sciences, College of Arts and Sciences, University of
the Philippines at Los Baños, Laguna 4031, Philippines.
tance to agriculture because it causes a stress
1
Present address: Environmental Resources Division, JIR- condition to crop plants. Particularly for rice
CAS, Tsukuba, Ibaraki 305, Japan. (Oryza sati6a L.), a species native to swamps and

0168-9452/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved.

PII S0168-9452(98)00025-9
2 M.L. Dionisio-Sese, S. Tobita / Plant Science 135 (1998) 1–9

freshwater marshes, secondary salinization is be- oxygen species in the mechanism of damage by
coming an increasingly serious production con- NaCl stress in rice plant, and also could allow
straint [1]. Because of the inherent sensitivity of deeper insights into the molecular mechanisms of
rice plant to salt stress [2], there has been a great tolerance to salt-induced oxidative stress.
interest in developing varieties that are resistant to
salinity. Defining salt tolerance, however, is quite
difficult because of the complex nature of salt 2. Materials and methods
stress and the wide range of plant responses.
One of the biochemical changes possibly occur- 2.1. Plant materials and salinity treatments
ring when plants are subjected to harmful stress
conditions is the production of activated oxygen Four rice varieties differing in salt tolerance
species. The chloroplasts and mitochondria of and being categorized into two ecogeographic lan-
plant cells are important intracellular generators draces were used. The japonica landraces were
of activated oxygen species. Electrons leaked from Hitomebore (salt-sensitive) and Bankat (salt-toler-
electron transport chains can react with O2 during ant) while the indica landraces comprised IR28
normal aerobic metabolism to produce activated (salt-sensitive) and Pokkali (salt-tolerant). Seeds
’
oxygen species such as superoxide (O2− ), hydro- of each variety were sown individually in holes of
gen peroxide (H2O2), and the hydroxyl radical a styrofoam board with a nylon net bottom (54
’
( OH). Singlet oxygen (1O2) can also be produced seeds of one genotype to an area of 200× 300 mm
through the energetic activation of ground state constituted a site). The styrofoam boards were
oxygen. These cytotoxic oxygen species are highly then floated on rectangular plastic trays half-filled
reactive and in the absence of any protective with 5 l of deionized water. They were then placed
mechanism they can seriously disrupt normal in a glasshouse under the natural light of a day/
metabolism through oxidative damage to lipids, night temperature maintained at 28°C. Seven and
protein and nucleic acids [3,4]. Fortunately plants 14 days after sowing, the deionized water was
possess a number of antioxidant enzymes that replaced, respectively, by one-fourth and one-half
protect them against the damaging effects of acti- strength modified Yoshida nutrient solution [5]
vated oxygen species. Superoxide dismutase adjusted to pH 5.8 with 1 N KOH. The nutrient
’
(SOD; EC1.15.1.1) is a major scavenger of O2− , solution was renewed twice a week and water lost
and its enzymatic action results in the formation by evapotranspiration was compensated for by
of H2O2 and O2. The hydrogen peroxide produced daily addition of deionized water. Three weeks
is then scavenged by catalase (EC 1.11.1.6) and a after sowing, salinization was induced by adding
variety of peroxidases (EC 1.11.1.7). Catalase, NaCl to the one-half strength modified Yoshida
which is apparently absent in the chloroplast, solution to obtain electrical conductivities of 6
dismutates H2O2 into water and molecular oxy- and 12 dS m − 1, which are equivalent to about 60
gen, whereas peroxidase decomposes H2O2 by ox- and 120 mM NaCl, respectively. Nutrient solution
idation of co-substrates such as phenolic without NaCl addition (0 mM NaCl) served as
compounds and/or antioxidants. the control, that is, the electrical conductivity was
This study was designed to determine, aside around 0 dS m − 1. Measurements were taken 7
from growth, the effect of salt stress on antioxi- days after salinity treatments. For antioxidant
dant enzyme activities, lipid membrane peroxida- enzyme determination and lipid peroxidation, leaf
tion, electrolyte leakage and Na + content of samples were harvested, weighed and stored at
leaves of rice varieties exhibiting differences in − 80°C prior to analysis. The electrolyte leakage
salinity tolerance. Comparison of these responses test was measured on freshly harvested leaf sam-
could be useful in identifying differences related ples while Na + determination was done on leaf
to the relative ability of each cultivar to cope with samples dried in an aerated oven at 80°C for 48 h.
salinity. Results from this study can supply infor- All analyses were done on a completely random-
mation on the possible involvement of activated ized design with four replicates. In all the figures
 M.L. Dionisio-Sese, S. Tobita / Plant Science 135 (1998) 1–9 3

the spread of values is shown as error bars repre- control and was deducted from A560. The reaction
senting standard errors of the means. mixture lacking enzyme developed the most color
and this decreased with increasing volume of ex-
2.2. Growth measurements tract added. Log A560 was plotted as a function of
the volume of enzyme extract in the reaction
Plants were randomly selected and gently up- mixture. The volume of enzyme extract producing
rooted to estimate growth by shoot dry weight 50% inhibition of the reaction was read from the
measurements. Dry weight was determined by resultant graph. The unit of SOD activity was
drying in an aerated oven at 80°C for 48 h. defined as that amount of enzyme which caused
Relative growth rate (RGR) was calculated from 50% inhibition of the initial rate of reaction in the
the increase in dry weight of plants at the begin- absence of enzyme.
ning and at the end of the salt treatment, using Peroxidase activity was determined using the
the equation RGR=(ln Wf −ln Wi)/(tf −ti) guaiacol oxidation method [8] in a 3 ml reaction
where W is the shoot dry weight, t is the time and mixture containing 10 mM phosphate buffer (pH
subscripts denote initial and final sampling, that 6.4), 8 mM guaiacol, 100–200 ml enzyme extract
is, 0 and 7 days after salinity treatment. and 2.75 mM H2O2. The increase in absorbance
was recorded at 470 nm within 30 s (linear phase)
2.3. Preparation of extracts after H2O2 was added [9]. A unit of peroxidase
activity was expressed as the change in ab-
For enzyme assays and estimation of lipid per- sorbance per minute and specific activity as en-
oxidation, frozen leaf samples were ground to a zyme units per mg soluble protein.
fine powder with liquid nitrogen and extracted
with ice-cold 50 mM phosphate buffer (pH 7.0). 2.5. Lipid peroxidation
The extracts were centrifuged at 4°C for 30 min at
20000× g and the resulting supernatants were Lipid peroxidation was determined by measur-
used as the crude extracts. ing the amount of malondialdehyde (MDA) for-
mation using the thiobarbituric acid method
2.4. Enzyme determinations described by Stewart and Bewley [7]. The crude
extract preparation was mixed with the same vol-
All spectrophotometric analyses were con- ume of a 0.5% (w/v) thiobarbituric acid solution
ducted at 25°C on a UV/visible light spectropho- containing 20% (w/v) trichloroacetic acid. The
tometer (U-2000, Hitachi, Tokyo, Japan). Total mixture was heated at 95°C for 30 min and the
SOD activity, the basis of which is its ability to reaction was stopped by quickly placing in an
inhibit the photochemical reduction of nitro blue ice-bath. The cooled mixture was centrifuged at
tetrazolium [6], was assayed following the proce- 10000× g for 10 min, and the absorbance of the
dure described by Stewart and Bewley [7]. A 3 ml supernatant at 532 and 600 nm was read. After
reaction mixture contained 50 mM phosphate subtracting the non-specific absorbance at 600
buffer (pH 7.8), 13 mM methionine, 75 mM nitro nm, the MDA concentration was determined by
blue tetrazolium, 2 mM riboflavin, 100 mM its extinction coefficient of 155 mM − 1 cm − 1.
EDTA, and 0–100 ml of enzyme extract. Ri-
boflavin was added last and the tubes were shaken 2.6. Electrolyte leakage
and placed 30 cm below two 15-W fluorescent
tubes giving a photon flux density of around 40 To determine electrolyte leakage, 100 mg fresh
mmol m − 2 s − 1. The reaction was allowed to run leaf samples were cut into 5 mm length and placed
for 10 min after which the lights were switched off in test tubes containing 10 ml distilled deionized
and the tubes covered with a black cloth. Ab- water. The tubes were covered with plastic caps
sorbance by the reaction mixture was read at 560 and placed in a water bath maintained at the
nm. The non-irradiated reaction mixture served as constant temperature of 32°C. After 2 h the initial
4 M.L. Dionisio-Sese, S. Tobita / Plant Science 135 (1998) 1–9

electrical conductivity of the medium (EC1) was 3.2. Effect of salinity on SOD and peroxidase
measured using an electrical conductivity meter acti6ities
(CM-115, Kyoto Electronics, Kyoto, Japan). The
samples were autoclaved afterwards at 121°C for Fig. 2 describes the effect of increasing level of
20 min to completely kill the tissues and release NaCl salinity on SOD activities of the four rice
all electrolytes. Samples were then cooled to 25°C varieties after 1-week exposure to salinity. The
and the final electrical conductivity (EC2) was leaves of the salt-sensitive varieties, Hitomebore
measured. The electrolyte leakage (EL) was ex- and IR28, exhibited a decline in SOD activity
pressed following the formula EL= EC1/EC2 × with increasing magnitude of salinity stress. The
100. moderately salt-tolerant japonica variety, Bankat,
showed a decrease in SOD activity at high salinity
2.7. Sodium determination level, whereas the salt-tolerant indica variety,
Pokkali, did not show any decline in SOD activity
For the determination of sodium in the leaf, 10 at all–in fact a slight increase in SOD activity can
mg dried material was cut into 1 cm length, be observed with increasing salinization.
placed in test tubes containing 20 ml distilled The peroxidase activity, on the other hand,
deionized water, and heated in a boiling water showed an opposite trend with regards to saliniza-
bath for 1 h. The tubes were then autoclaved at tion in all the varieties tested (Fig. 3). Unlike
121°C for 20 min and cooled. The sodium content SOD, peroxidase activity increased with increas-
in 15 times diluted extract was determined by ing salinity level in Hitomebore and IR28 vari-
atomic absorption spectrophotometry (AA-660, eties. The moderately salt-tolerant Bankat also
Shimadzu, Japan). showed an increasing peroxidase activity with in-
creasing salinization similar to the sensitive vari-
2.8. Protein determination eties. A slight decrease in peroxidase activity,
however, was observed in the tolerant Pokkali
Soluble protein content of the extracts was upon increasing exposure to salinity stress.
determined according to the method of Bradford
[10] using the Bio-Rad assay kit with bovine
serum albumin as a calibration standard.

3. Results

3.1. Effect of salinity on growth

Based on shoot dry weight, the relative growth

rates of the four O. sati6a varieties subjected to
1-week salinity treatments are shown in Fig. 1.
Varieties which are considered as salt-tolerant,
that is, Bankat and Pokkali, showed higher
growth rate values at 6 dS m − 1 salinity level
compared to the non-salt-treated plants. The salt-
sensitive varieties, Hitomebore and IR28, on the
other hand, did not show this growth stimulation
at moderate salinity level. At 12 dS m − 1 salinity
level, all of the tested varieties including Bankat,
Fig. 1. Effect of increasing salinity on relative growth rates in
except Pokkali, showed growth retardation, terms of shoot dry weight of the four rice varieties. Values are
reflecting the greater salt tolerance of Pokkali means from four replications and bars indicate standard er-
compared to Bankat. rors.
 M.L. Dionisio-Sese, S. Tobita / Plant Science 135 (1998) 1–9 5

Fig. 2. Effect of increasing salinity on the activity of SOD in Fig. 4. Effect of increasing salinity on MDA content in the
the leaves of four rice varieties. leaves of four rice varieties.

3.3. Effect of salinity on lipid peroxidation shown in Fig. 4. With increasing level of salinity
stress, the MDA content increased in the sensitive
The effect of increasing magnitude of salinity varieties including Bankat, thus indicating an in-
stress on MDA formation in the leaves of the four crease in lipid peroxidation. Pokkali, on the other
rice varieties after 1-week salinity treatment is hand, did not exhibit this increase in lipid peroxi-
dation with a 1-week exposure to salinity stress.

3.4. Effect of salinity on electrolyte leakage

The extent of membrane damage was assessed

indirectly by conductometric measurements of so-
lute leakage from cells. The amount of electrolyte
leakage from the leaves of the four rice varieties
subjected to increasing level of salinity stress is
shown in Fig. 5. The results from both the salt-
sensitive varieties were very similar. Increasing
magnitude of salinity stress increased the amount
of electrolyte leakage from the leaves of Hitome-
bore and IR28. The Bankat variety showed a
trend similar to the sensitive varieties while the
rate of electrolyte leakage was almost unchanged
in Pokkali.

3.5. Effect of salinity on Na + uptake

Fig. 3. Effect of increasing salinity of the activity of peroxidase
in the leaves of four rice varieties. There was a marked varietal difference in the
6 M.L. Dionisio-Sese, S. Tobita / Plant Science 135 (1998) 1–9

accumulation of Na + in rice leaves in response to

salinity. The salt-sensitive varieties, Hitomebore
and IR28, as well as Bankat showed increasing
Na + content with increasing salinization while
Pokkali did not exhibit this pronounced accumu-
lation of Na + during 1-week exposure to salinity
stress (Fig. 6).

4. Discussion

Considerable effort has been exerted in the

selection and development of rice varieties resis-
tant to salinity stress. Progress, however, seems
slow primarily due to inadequate understanding
of the mechanism of salt tolerance.
In rice, as well as in other crops, agronomic
characteristics, such as survival, yield, leaf dam-
age and plant height are the most commonly
Fig. 6. Effect of increasing salinity on Na + accumulation in
used criteria for measuring salt tolerance [11,12].
the leaves of the four rice varieties.
This is probably due to their ease of measure-
ment and the fact that yield under saline condi-
tions, is what ultimately matters. It has been clusion, are more relevant criteria for improving
proposed, however, that physiological mecha- salt tolerance in crops [13].
nisms underlying salt tolerance, such as ion ex- Results of the analysis of Na + content of
leaves of rice varieties differing in salt tolerance
agree with the view that there is an inverse rela-
tionship between shoot Na + concentration and
salt tolerance [14]. The salt-tolerant variety Pok-
kali did not exhibit a significant increase in Na +
accumulation in the leaves even at high salinity
level whereas the salt-sensitive varieties Hitome-
bore and IR28 showed pronounced accumula-
tion (Fig. 6). Interestingly, the putative
salt-tolerant variety Bankat exhibited Na + accu-
mulation under salinity stress similar to the salt-
sensitive varieties. These distinct susceptibilities
of rice varieties to shoot Na + accumulation may
have brought about the differential responses in
the activities of antioxidant enzymes involved in
oxygen metabolism during salt stress.
Salinity caused a significant decrease of SOD
activity in the salt-sensitive varieties as well as in
Bankat while in the salt-tolerant variety Pokkali,
SOD activity slightly increased in response to
Fig. 5. Effect of increasing salinity on electrolyte leakage rate salt treatment (Fig. 2). Singha and Choudhuri
of the leaves of the four rice varieties. [15] also observed that NaCl decreased SOD act-
 M.L. Dionisio-Sese, S. Tobita / Plant Science 135 (1998) 1–9 7

ivity in rice seedlings. Since they did not report was due to increased activity of peroxidase encod-
differential varietal salt sensitivity, it is probably ing genes or increased activation of already exist-
that they used salt-sensitive varieties. ing enzymes. Mittal and Dubey [21] suggested
Salinity also decreased SOD activity in the that salinity affects mainly the de novo synthesis
leaves, chloroplasts and mitochondria of pea of the enzyme since inhibition in vitro conditions
plants [16,17]. Hernandez et al. [18] reported that and activation in vivo conditions were observed in
the catalytic activity of SOD isozymes from salt-sensitive cultivars. Lopez et al. [24], however,
cowpea plants decreased as a function of salt has shown that the salt-induced increase in ascor-
concentration in vitro. Although the compartmen- bate peroxidase activity in radish plants was not
tation of Na + within leaves and inside the cells accompanied by a corresponding increase in
was not determined in the present study, the high mRNA level, suggesting that the salt-induced
ion content and the parallel decrease in SOD ascorbate peroxidase expression is probably the
activities found in the leaves of salinized rice consequence of post-transcriptional events.
plants is consistent with the possibility that salt Aside from their function in the metabolism of
directly inhibits the catalysis in vivo. However, active oxygen, peroxidases in plants are also in-
the other possibility that the inhibition of SOD volved in the biosynthesis of cell wall [25] includ-
activity under salt stress is a consequence of an ing lignification and suberization [26,27].
altered synthesis and accumulation of less active Considerable evidence shows that high peroxidase
enzymes in salt-treated plants cannot be entirely activity is correlated with the reduction of plant
ruled out. growth [9,28,29]. This might be attributed to per-
The observed decrease in SOD activity in salin-
oxidase catalysis of ferulic acid conversion to
ized salt-sensitive rice varieties, including Bankat,
diferulic acid on polysaccharides, the feruloyla-
in turn, could diminish the ability of the seedlings
’ tion of hemicelluloses, or the insolubilization of
to scavenge O2− radicals favoring an accumula-
hydroxyproline-rich glycoprotein causing cell wall
tion of oxygen radical species, which could cause
stiffening [30–32]. Morphologically, the most typ-
membrane damage. The extent of damage to the
ical symptom of saline injury to a plant is re-
membrane was monitored by measuring the
tarded growth due to inhibition of cell elongation
amount of thiobarbituric-acid-reactive material
(MDA) produced when polyunsaturated fatty [33], resulting in a stunted plant. Notwithstanding
acids in the membrane undergo peroxidation. Un- the other physiological and biochemical mecha-
like the salt-tolerant Pokkali, there was an ob- nisms involved, the observed decrease in rice
served increase of MDA in all the varieties tested growth of salt-sensitive varieties including Bankat
when exposed to NaCl stress (Fig. 4). By generat- under high salinity level might then be partly due
ing changes in unsaturated fatty acids that affect to salt-induced increases in peroxidase activity.
membrane structure and properties, this enhanced The results of this study show that there were
free radical formation and lipid peroxidation un- substantial differences between the growth and
der salt stress in salt-sensitive varieties may have antioxidant responses of the four rice varieties to
also brought about an increase in membrane per- salinity treatment. During salt stress the known
meability or loss of membrane integrity, as evi- salt-sensitive varieties, Hitomebore and IR28, ex-
denced by the increase in solute leakage (Fig. 5). hibited high leaf Na + accumulation resulting in
Salt stress-induced electrolyte leakage has also symptoms of oxidative damage such as decrease
been previously observed in tomatoes [19] and in SOD activity, increase in lipid peroxidation and
melons [20]. electrolyte leakage, increase in peroxidase activity
Various researchers dealing with rice [21] and and decrease in growth rate. Salinity however,
other plants [22,23] have also reported increase in only had a minimal effect on leaf Na + accumula-
peroxidase activity in salt-sensitive cultivars under tion and antioxidant metabolism in the known
salt stress. It is not clear whether the observed salt-tolerant variety, Pokkali, and its growth rate
increase in peroxidase activity under salt stress slightly enhanced at moderate salinity level. The
8 M.L. Dionisio-Sese, S. Tobita / Plant Science 135 (1998) 1–9

putative salt tolerant Bankat variety however, [8] B. Chance, C. Maehly, Assay of catalase and peroxidases,
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The authors wish to thank Drs Takaharu
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