RESEARCH ARTICLE

Production and detoxification of H2O2 in lettuce plantsexposed to seleniumJ.J. Rıos, B. Blasco, L.M. Cervilla, M.A. Rosales, E. Sanchez-Rodriguez, L. Romero & J.M. Ruiz

Department of Plant Physiology, Faculty of Science, University of Granada, Granada, Spain

Keywords

Antioxidant enzymes; hydrogen peroxide;

Lactuca sativa; lipid peroxidation; selenate;

selenite.

Correspondence

J.J. Rıos, Department of Plant Physiology,

Faculty of Science, University of Granada. Avd.

Fuente Nueva s/n, E-18071 Granada, Spain.

Email: jjrios@ugr.es

Received: 5 March 2008; revised version

accepted: 28 May 2008.

doi:10.1111/j.1744-7348.2008.00276.x

Abstract

Selenium is considered an essential element for animals. Despite that it has not

been demonstrated to be essential for higher plants, it has been attributed with

a protective role against reactive oxygen species in plants subjected to stress. In

this study, lettuce plants (Lactuca sativa cv. Philipus) received different applica-

tion rates (5, 10, 20, 40, 60, 80 and 120 lM) of selenite or selenate, with the

aim of testing the effect of Se on the production and detoxification of H2O2 in

non-stressed plants. The results indicate that the form selenate is less toxic

than selenite; that is, the plants tolerated and responded positively to this

element, and even increasing in growth up to a rate of 40 lM for the form

selenate. On the contrary, the application of selenite triggered a higher

foliar concentration of H2O2 and a higher induction of lipid peroxidation

[malondialdehyde content and lipoxygenase activity] in comparison to that

observed after the selenate application. Also, the plants treated with selenate

induced higher increases in enzymes that detoxify H2O2, especially ascorbate

peroxidase and glutathione (GSH) peroxidase, as well as an increase in the

foliar concentration of antioxidant compounds such as ascorbate and GSH.

These data indicate that an application of selenate at low rates can be used to

prevent the induction in plants of the antioxidant system, thereby improving

stress resistance.

Introduction

Selenium has been considered essential to animal nutri-

tion since 1957, and humans have a daily requirement of

50–70 lg day21 (U.S. Department of Agriculture, 2003)

as a component of the enzymes glutathione peroxidase

(GSH-Px), selenoprotein and tetraiodothyronine 5#-de-iodinase. The studies have recommended a dietary Se

supplement of 100–200 lg day21 to decrease the inci-

dence of lung and prostate cancer (Ip et al., 1991; Ip &

Ganther, 1992; Lauchli, 1993; Clark et al., 1996). Fur-

thermore, although Se is not considered to be required

by higher plants, there are indications that it exerts posi-

tive effects on plants. Recent research has identified sev-

eral beneficial effects of Se in plants which include

antioxidant properties that can stimulate plant growth

(Hartikainen et al., 2000), delay plant senescence

(Djanaguiraman et al., 2005), protect plants from

phloem-feeding aphids by means of deterrence and tox-

icity to aphids (Hanson et al., 2004), protect against fun-

gal infection and from herbivory (Hanson et al., 2003)

and protect plants against different types of abiotic stress

(Hartikainen & Xue, 1999).

The beneficial effect of Se in plants subjected to stress

conditions has in most cases been attributed to increased

antioxidant activity. Thus, Xue et al. (2001) and after-

wards Djanaguiraman et al. (2005) observed the effect of

Se application in the form of selenate on senescence in

lettuce and soy, confirming that the decline in antioxi-

dant enzyme activity was milder in plants treated with

this element, which offsets oxidative damage by boosting

growth in plants treated with Se.

Annals of Applied Biology ISSN 0003-4746

Ann Appl Biol 154 (2009) 107–116 ª 2008 The Authors

Journal compilation ª 2008 Association of Applied Biologists

107

Kong et al. (2005), studying the effect of applying sele-

nate to sorrel plants under saline stress, reported an

increase in antioxidant enzymes and furthermore an

improvement in the integrity of plasma, mitochondrial

and chloroplast membranes. These data, therefore, apart

from suggesting an antioxidant function of Se, define

this element as a strengthener of osmotic capacity for its

role in the maintenance of cell membranes.

Recently, a beneficial effect of Se on heavy metal toxic-

ity has been reported. Pedrero et al. (2008) studied the

response of broccoli submitted to Cd toxicity together

with the application of Se. This study reported that Se

application diminished the malondialdehyde (MDA)

content and decreased the translocation of Cd towards

the shoot, thereby reducing oxidative stress provoked by

this heavy metal.

The greater antioxidant activity after Se application has

been related by several authors to its possible action on the

enzyme GSH-Px. Although the existence of Se-dependent

GSH-Px has not been demonstrated in plants, as opposed

to the situation in animals, it appears that this enzyme

could be influenced by the presence of Se. Support of

this idea comes from several works such as that of

Shigeoka et al. (1991), who observed after purifying the

GSH-Px in the alga Chlamydomonas that it was very simi-

lar to the one characterised in humans. Afterwards,

Takeda et al. (1997) demonstrated in this same alga that

H2O2 was eliminated primarily by the enzyme ascorbate

peroxidase (APX) in the absence of Se; however, when

this trace element was applied, the H2O2 was detoxified

mainly by GSH-Px. Afterwards, work on higher plants,

such as those by Hartikainen et al. (1997, 2000), credit

Se with antioxidant properties at low concentrations for

the inducing power of GSH-Px.

However, apart from GSH-Px, other enzymes such as

superoxide dismutase (SOD) form an integral part in oxi-

dative metabolism and function to transform the radical

superoxide into H2O2 (Gratao et al., 2005). In this res-

pect, Kong et al. (2005), in sorrel plants under saline

stress found stronger SOD activity. On the contrary, in

the same work, the authors failed to find the same behav-

iour for the enzyme catalase (CAT), which was not af-

fected by the presence or absence of Se.

In short, the effect of Se in promoting antioxidant activ-

ity has been reported basically in plants subjected to any

type of stress, while the possible action of this trace ele-

ment (different application rates as well as forms selenate

versus selenite) in the oxidative metabolism of non-

stressed plants has hardly been documented. Notable

among the few works on this subject, the study by

Cartes et al. (2005) reported that lipid peroxidation de-

pended on the shoot Se concentration rather than the Se

source (selenite or selenate) in ryegrass plants. In this

study, the shoot Se concentration was significantly

greater in selenate than in selenite-treated plants, and

the pro-oxidant effect of Se was detected only in plants

treated with selenate. Recently, Gomes-Junior et al.

(2007) found that in coffee cell suspensions the applica-

tion of selenite at low concentrations (0.05 mM) pro-

duces lipid peroxidation and alterations in antioxidant

enzymes including a severe reduction in APX activity.

These types ofwork are of great importance at present in

the sense that Se is vital to mammalian nutrition because

Se deficiency may promote cancer (Diwadkar-Navsar-

iwala et al., 2006). Dietary Se deficiency in humans is

caused by the ingestion of plant foods with an impercep-

tible concentration of this element because of its low

bioavailability in most crop soils (Smorklji et al., 2005;

Pedrero et al., 2006). Therefore, as a result of this low

bioavailability and the role of plants as the main dietary

source of this element, more studies have appeared in

recent years concerning ways to increase the Se content

in plants used for human consumption through bio-

fortification programmes (Rıos et al., 2008). Thus, the

main aim of the present work was to determine, in non-

stressed plants, the way in which Se applied at different

rates and in different forms (selenate vs. selenite) affects

the formation and detoxification of H2O2.

Material and methods

Plant material and growing conditions

Seeds of Lactuca sativa L. cv. Philipus were germinated

and grown for 35 days in cell flats (cell size, 3 � 3 � 10

cm) filled with perlite mixture, and the flats were placed

on benches in an experimental greenhouse in southern

Spain (Saliplant S.L., Motril, Granada, Spain). The 35-

day-old seedlings were transferred to a cultivation cham-

ber under controlled environmental conditions with

relative humidity of 60–80%, temperature 25�C/15�C(day/night) and 12/12 h photoperiod at a photosynthetic

photon-flux density (PPFD) of 350 lmol m22 s21 (mea-

sured at the top of plants with a 190 SB quantum sensor;

LI-COR Inc., Lincoln, NE, USA). The plants were grown

in individual pots (25 cm upper diameter, 17 cm lower

diameter, 25 cm in height) of 8 L volume, filled with ver-

miculite. Until the end of experiment, the plants received

a growth solution composed of 4 mM Ca(NO3)2, 6 mM

KNO3, 2 mM MgSO4�7H2O, 1 mM NaH2PO4�2H2O, 50 lMH3BO3, 2 lM MnCl2�4H2O, 1 lM ZnSO4�7H2O, 0.1 lMNa2MoO4�2H2O, 0.25 lM CuSO4�5H2O and 10 lM Fe-ED-

DHA. The nutrient solution (pH 5.5–6.0) was renewed

every 3 days, and the vermiculite partly rinsed with

Millipore-filtered water to avoid nutrient accumulation.

Selenium and antioxidant system in lettuce plants J.J. Rıos et al.

108 Ann Appl Biol 154 (2009) 107–116 ª 2008 The Authors

Journal compilation ª 2008 Association of Applied Biologists

At 45 days after germination, the different Se dosages

were applied together with the nutrient solution de-

scribed above and maintained to the end of the experiment

(for 21 days). The concentrations of Se added as SeO4 =

(Na2SeO4) or SeO3 = (Na2SeO3) were 5, 10, 20, 40, 60, 80

and 120 lmol L21. The choice of the different Se ap-

plication rates was to cover a broad range, from the rec-

ommended rate for biofortification programmes to rates

causing phytotoxicity (Rıos et al., 2008). In addition to

these treatments, a control treatment consisted of ap-

plying the complete growth solution without a Se sup-

plement. The experimental design was a randomised

complete block with 15 treatments, arranged in individ-

ual pots with six plants per treatment and three repli-

cations. The experiment was repeated three times under

the same conditions (n = 9).

Plant sampling

Lettuce leaves were sampled on day 66 after sowing. Leaf

samples were standardised using only fully expanded

leaves from the middle part of plants in each replicate, as

these reflect most clearly, from the nutritional and meta-

bolic standpoint, the effect of the treatment applied. The

material was rinsed three times in distilled water after dis-

infection with non-ionic 1% detergent and then blotted

onfilter paper. At each sampling, freshmatterwas used for

analysis of enzyme activity: lipoxygenase (LOX), SOD,

CAT, APX and GSH-Px; MDA, H2O2, ascorbate and

glutathione (GSH) content. The rest of the plant material

(edible leaves) was lyophilised and used to determine

the biomass and Se concentration.

Plant analysis

Measurement of lipid peroxidation

For the assay of MDA, 0.5 g of lettuce leaf was homoge-

nised in 5 mL of 50 mM buffer solution (containing

0.07% of NaH2PO4�2H2O and 1.6% Na2HPO4�12H2O),

ground with a mortar and pestle on ice, and centrifuged

at 20 000 g for 25 min (4�C). The MDA concentration

was calculated using its extinction coefficient of

155 mM21 cm21 (Fu & Huang, 2001). LOX activity was

measured according to Minguez-Mosquera et al. (1993),

using 50 mM K-phosphate buffer (pH 6.0) for extrac-

tion. The LOX activity was calculated following the rise

in the extinction at A234 using an extinction coefficient

of 25 000 M21 cm21 (Egert & Tevini, 2002).

H2O2 concentration

The H2O2 content of leaf samples was colorimetrically

measured as described by Mukherjee & Choudhuri

(1983). Leaf samples were extracted with cold acetone

to determine the H2O2 levels. An aliquot (1 mL) of the

extract solution was mixed with 200 lL of 0.1% titan-

ium dioxide in 20% (v/v) H2SO4 and the mixture was

then centrifuged at 6000 g for 15 min. The intensity of

the yellow colour of the supernatant was measured at

415 nm. The H2O2 concentration was calculated from

a standard curve plotted within the range of 100–

1000 nmol H2O2.

Measurement of total ascorbate and glutathione content

The total ascorbate was determined spectrophotometri-

cally following Gossett et al. (1994). Lettuce leaves [1–

1.5 g fresh weight (FW)] were homogenised over ice

with 0.5 g inert sand and 10 mL of ice-cold 5% (w/v)

m-phosphoric acid freshly prepared with a mortar and

a pestle. The homogenate was centrifuged at 21 800 g

for 15 min at 2�C. Total ascorbate was determined by

incubating at 25�C for 30 min of a reaction mixture con-

sisting of 100 lL supernatant, 500 lL 110 mM KH2PO4,

100 lL 3.6 mM ethylenediaminetetraacetic acid (EDTA)

and 100 lL 1.5 mM dithiothreitol (DTT), this latter

compound applied to reduce all dehydroascorbate and

ascorbate to avoid ascorbate oxidation. After incuba-

tion, 100 lL of 0.5% (w/v) N-ethylmaleimide was

added to remove excess DTT. Colour was developed

by the addition of 400 lL 10% (w/v) trichloroacetic

acid, 400 lL 44% o-phosphoric acid, 400 lL of 65 mM

a-a-1-dipyridyl in 70% ethanol and 200 lL of 110 mM

FeCl3. The reaction mixture was then incubated at 37�Cfor 45 min in a water bath and quantified at 525 nm.

Total GSH was determined following Gossett et al.

(1994). The extraction was made by homogenisation in

metaphosphoric acid 5% (w/v). The homogenate was

centrifuged at 21 800 g for 15 min. Afterwards, the reac-

tion mixture was made of 50 lL extract, 250 lL buffer,

HEPES-HCl 50 mM (pH 7.6) that contained 330 mM

betaine and 150 lL sulphosalicylic acid at 10 % (v/v).

Afterwards, in a test tube, 150 lL of the above reaction

mixture, 700 lL of NADPH 0.3 mM, 100 lL of 5,5#-dithiobis-(2-nitrobenzoic acid) (DNTB) 6 mM and 50 lLof glutathione reductase (10 U mL21) were added.

Finally, the absorbances of the samples were read at

412 nm. A standard curve was drawn by preparing sol-

utions of 0.002–0.0001 g mL21 GSH in 60 mL meta-

phosphoric acid (pH 2.8) containing 1 mM EDTA and

determining in the same manner as for the extracts.

Activity of antioxidant enzymes

Superoxide dismutase (EC 1.15.1.1) activity was assayed

by monitoring the inhibition of the photochemical

J.J. Rıos et al. Selenium and antioxidant system in lettuce plants

Ann Appl Biol 154 (2009) 107–116 ª 2008 The Authors

Journal compilation ª 2008 Association of Applied Biologists

109

reduction of nitroblue tetrazolium (NBT), according to

the methods of Giannopolitis & Ries (1977) and Beyer &

Fridovich (1987), with some modifications. A 5-mL reac-

tion mixture was used, containing 50 mM Na2CO3 (pH

10.0), 13 mM methionine, 0.025% (v/v) Triton X-100,

63 lM NBT, 1.3 lM riboflavin and an appropriate aliquot

of enzyme extract. The reaction mixtures were illumi-

nated for 15 min at a PPFD of 380 lmol m22 s21. Mix-

tures not illuminated were used to correct for background

absorbance. One unit of SOD activity was defined as the

amount of enzyme required to cause 50% inhibition of

the reduction of NBT as monitored at 560 nm.

Catalase (EC 1.11.1.6) activity was determined by fol-

lowing the consumption of H2O2 at 240 nm for 5 min

(Nakano & Asada, 1981; Rao et al., 1997). The reaction

mixture (3 mL total volume) contained 25 mM Tris–

acetate buffer (pH 7.0), 0.8 mM EDTA-Na and 20 mM

H2O2, and the enzyme assay was performed at 25�C.The enzyme APX (EC 1.11.1.11) was assayed following

Rao et al. (1996). APX activity was determined by register-

ing the absorbance change at 290 nm for 3 min of a reac-

tion mixture (3.75 mL) containing 100 mM of potassium

phosphate buffer (pH 7.5), 0.5 mM of reduced ascorbate,

0.2 mM of H2O2 and 0.75 mL of enzyme extract.

The GSH-Px (EC 1.11.1.9) activity was measured by

a modification of the method of Flohe & Gunzler (1984)

using H2O2 as substrate. The enzyme was extracted by

prechilled KNaHPO4 buffer, pH 7.0, with homogeniser.

The supernatant obtained by centrifugation at 1100 g for

10 min was used as a coarse enzyme extract. For the

enzyme reaction, 0.2 mL of the supernatant was placed

in tube and mixed with 0.4 mL GSH (0.1 mM) and

0.2 mL KNaHPO4 (0.067 M). The above reagents with-

out supernatant extract were used for the non-enzyme

reaction. After preheating the mixture on water bath at

25�C for 5 min, 0.2 mL H2O2 (1.3 mM) was added to

initiate the reaction. The reaction lasted 10 min and was

stopped by adding 1 mL 1% trichloroacetic acid and the

mixture was put into an ice bath for 30 min. Then the

mixture was centrifuged for 10 min at 1100 g, 0.48 mL

the supernatant was placed into a cuvette and 2.2 mL of

0.32 M Na2HPO4 and 0.32 mL of 1.0 mM DNTB were

added for colour development. The absorbance at wave-

length 412 nm was measured after 5 min. The enzyme

activity was calculated as a decrease in GSH within the

reaction time when compared with that in the non-

enzyme reaction.

Measurement of total Se content

For the determination of the Se concentration, 25 mg of

sample were digested with 2.5 mL of concentrated HNO3

and 1 mL of H2O2 in an analytical microwave oven. The

resulting solution was diluted to 25 mL with deionised

water and the metal concentration determined by induc-

tively coupled plasma mass spectroscopy (ICP-MS) ac-

cording to Pedrero et al. (2006).

Statistical analysis

The data were subjected to a simple ANOVA at 95% con-

fidence, using the program Statgraphics 6.1. A two-way

ANOVA was applied to ascertain whether the Se appli-

cation rate, and forms applied, significantly affected the

results, and the means were compared by Fisher’s least

significant differences (LSD). The data shown are mean

values � standard errors (SE). The significance levels for

both analyses were expressed as: *P < 0.05; **P < 0.01;

***P < 0.001 and ns, not significant.

Results

Selenium, despite not being an essential element for

plants, is absorbed and accumulated by them depending

on the application rate and the form available in the

culture medium. As expected, foliar Se concentration

increased with increasing application rate (P < 0.001),

peaking in the 120 lM treatment for both Se forms

applied (Fig. 1). It is striking that for most of the applica-

tion rates, the foliar concentration of Se was higher with

selenate than with selenite (Fig. 1).

In relation to the shoot biomass, the highest valueswere

registered at the rate of 20 lMfor selenate and 5 lMin the

case of selenite. These concentrations resulted in greater

plant growth than in control (Table 1). Finally, plant

growth diminished to a concentration of 120 lM where

lower shoot biomass production was found for both Se

forms applied (Table 1).

With respect to stress indicators used in this experiment,

we found that the external application of the different

rates of Se augmented LOX activity, foliar concentration

of MDA, with the treatment of 120 lM showing the high-

est values, and the control plants the lowest (Table 1). As

for these parameters, at all rates selenite application

induced higher values than did selenate (Table 1). In

addition, Table 1 shows an increase in the H2O2 concen-

tration as the Se application rate increases, its concentra-

tion being again higher when the form used was

selenite. The dissimilar effect of the two Se forms on the

H2O2 concentration is striking because, when selenite

was applied at a concentration of 20 lM, the H2O2 con-

centration rose significantly with respect to control,

while for selenate this did not occur until at the concen-

tration of 120 lM (Table 1).

During oxidative stress, the excess reactive oxygen spe-

cies (ROS) production causes membrane damage that

Selenium and antioxidant system in lettuce plants J.J. Rıos et al.

110 Ann Appl Biol 154 (2009) 107–116 ª 2008 The Authors

Journal compilation ª 2008 Association of Applied Biologists

eventually leads to cell death. For protection against ROS,

plants contain antioxidant enzymes such as SOD, CAT,

APX andGSH-Px, aswell as awide array of non-enzymatic

antioxidants (Mittler, 2002; Blokhina et al., 2003).

Table 2 shows the dissimilar responses found for the

enzymatic activities related both to the formation and to

the detoxification of H2O2. The activity of SOD increased

with increasing Se application rate, reaching maximum

values in the treatments 40 lM of selenite and 80–

120 lM of selenate (Table 2). In relation to CAT, the re-

sults indicate that there were no significant differences in

its activity with respect to the Se application rate (Table 2).

The activity of APX was higher when selenate was

applied, the activity being highest at the rate of 20 lM and

the lowest at 120 lM, while in the rest of the treatments

the APX activity surpassed control values (Table 2). On

the other hand, when selenite was added, only the 5 lMconcentration presented a value higher than control, re-

flecting the lower activity at the concentrations of 80 and

120 lM (Table 2). On the contrary, the GSH-Px activity

increased as the Se rate rose, this increase being higher

when the form applied was selenate (Table 2).

Finally, with respect to ascorbate and GSH our results

indicate for both compounds that the application of

0

5

10

15

20

25

30

35

40

45

50

0 5 10 20 40 60 80 120

µmol L-1

mg

Se

Kg

-1 D

W

Selenate LSD0.05 0.40Selenite LSD0.05 0.70

Figure 1 Foliar concentration of Se found in lettuce plants subjected to different concentrations and forms of Se. Data are means � SE (n = 9).

Table 1 Shoot biomass, lipid peroxidation (MDA content and LOX activity) and H2O2 concentration in lettuce plants subject to different concentration

and forms of Se [selenate: Se(VI); selenite: Se(IV)]a

Treatments Shoot Biomass (g DW)

LOX Activity

(lmol min21 mg21 protein) MDA Content (lmol g21) H2O2 Concentration (lmol g21 FW)

Doses Se (lM) Se(IV) Se(VI) Se(IV) Se(VI) Se(IV) Se(VI) Se(IV) Se(VI)

0 2.38 � 0.16 2.38 � 0.16 0.21 � 0.03 0.21 � 0.03 6.88 � 0.13 6.88 � 0.13 0.84 � 0.07 0.84 � 0.07

5 2.62 � 0.18 2.41 � 0.18 0.30 � 0.03 0.22 � 0.02 7.51 � 0.17 7.16 � 0.19 0.72 � 0.07 0.81 � 0.06

10 2.18 � 0.15 2.45 � 0.16 0.32 � 0.04 0.26 � 0.03 8.03 � 0.16 7.26 � 0.25 0.86 � 0.06 0.81 � 0.07

20 1.79 � 0.12 2.61 � 0.15 0.35 � 0.03 0.30 � 0.03 8.97 � 0.22 7.34 � 0.20 1.09 � 0.09 0.80 � 0.09

40 1.69 � 0.11 2.43 � 0.17 0.36 � 0.02 0.33 � 0.02 9.49 � 0.20 7.55 � 0.22 1.03 � 0.08 0.75 � 0.08

60 1.69 � 0.11 2.40 � 0.16 0.38 � 0.03 0.34 � 0.04 10.7 � 0.19 7.98 � 0.27 1.23 � 0.09 0.86 � 0.07

80 1.40 � 0.09 2.26 � 0.15 0.38 � 0.04 0.35 � 0.05 11.0 � 0.27 8.05 � 0.31 1.39 � 0.12 0.88 � 0.07

120 1.35 � 0.08 2.09 � 0.13 0.42 � 0.05 0.37 � 0.04 11.9 � 0.25 8.12 � 0.29 1.12 � 0.11 0.97 � 0.08

P-value *** *** *** ** *** *** *** *

LSD 0.21 0.12 0.01 0.08 0.06 0.14 0.12 0.10

Analysis of variance

Forms Se (F) *** *** *** ***

Doses Se (D) *** *** *** ***

F � D *** NS *** ***

LSD 0.16 0.04 0.07 0.08

FW, fresh weight; LOX, lipoxygenase; LSD, least significant differences; MDA, malondialdehyde.aData are means � SE (n = 9). Levels of significance are represented by

*P < 0.05, **P < 0.01, ***P < 0.001 and NS, not significant.

J.J. Rıos et al. Selenium and antioxidant system in lettuce plants

Ann Appl Biol 154 (2009) 107–116 ª 2008 The Authors

Journal compilation ª 2008 Association of Applied Biologists

111

selenate in general more strongly induced the synthesis

and accumulation of these antioxidant compounds

(Fig. 2, P < 0.001 and Fig. 3, P < 0.001, respectively).

Discussion

Foliar Se concentration increaseswith increasing application

rate, and this was higher with selenate than with selenite

(Fig. 1). These data coincide with those reported in works

conducted with different plants, showing a higher Se con-

centration with greater application rates (Hartikainen

et al., 1997; Cartes et al., 2005). In relation to the Se form

applied, our results are similar to those of Hopper & Parker

(1999), who observed that the application of selenate

implies a translocation of Se towards the leaves greater

than or equal to 84%, whereas the selenite application

meant Se translocation of less than or equal to 47%.

In plants, stress negatively affects growth and develop-

ment and also generates ROS that damagenumerousmacro-

molecules and cell structures. Consequently, under adverse

conditions, some of the most reliable and widely used indi-

cators of stress are plant biomass and lipid peroxidation

expressed as LOX activity and MDA concentration (Fu &

Huang, 2001; Sairam et al., 2002; Rosales et al., 2006).

Therefore, oneway of defining the effect of the different

Se treatments in our experiment was to study these indi-

cators of oxidative stress. Our results with respect to shoot

biomass reveal significant differences depending on the

Table 2 Activity of the enzymes in charge of the formation and detoxification of H2O2 in lettuce plants subjected to different concentrations and forms

of Se [selenate: Se(VI); selenite: Se(IV)]a

Treatments SOD (U min21 g21 FW)

CAT

(lmol H2O2 min21 g21 FW)

APX

(nmol NADPH min21 g21 FW)

GSH-Px

(nmol GSH min21 g21 FW)

Doses Se (lM) Se(IV) Se(VI) Se(IV) Se(VI) Se(IV) Se(VI) Se(IV) Se(VI)

0 1.70 � 0.13 1.70 � 0.13 21.22 � 0.34 21.22 � 0.34 101.54 � 2.57 101.54 � 2.57 0.93 � 0.07 0.93 � 0.07

5 1.82 � 0.12 1.46 � 0.10 17.45 � 0.31 20.84 � 0.30 125.91 � 3.17 119.61 � 2.63 2.45 � 0.16 1.36 � 0.11

10 1.80 � 0.13 1.88 � 0.12 18.54 � 0.31 20.13 � 0.32 91.54 � 2.28 129.57 � 2.77 2.17 � 0.18 1.46 � 0.13

20 2.15 � 0.18 1.77 � 0.12 20.03 � 0.33 29.23 � 0.37 90.08 � 2.65 146.67 � 3.24 2.62 � 0.15 3.08 � 0.22

40 2.73 � 0.20 1.76 � 0.11 17.60 � 0.27 28.14 � 0.36 79.33 � 1.89 143.75 � 3.51 2.96 � 0.17 2.62 � 0.18

60 2.07 � 0.20 1.66 � 0.10 22.77 � 0.28 26.80 � 0.39 74.58 � 2.07 136.08 � 3.44 3.51 � 0.23 3.14 � 0.24

80 2.49 � 0.19 1.97 � 0.15 21.52 � 0.29 26.66 � 0.38 67.12 � 1.78 135.75 � 2.97 3.99 � 0.27 5.39 � 0.37

120 2.37 � 0.21 2.14 � 0.17 20.74 � 0.32 27.55 � 0.37 69.97 � 2.01 90.35 � 2.85 4.81 � 0.32 5.47 � 0.41

P-value *** ** NS NS *** *** *** ***

LSD 0.20 0.23 8.74 9.21 17.17 19.19 0.89 0.29

Analysis of variance

Forms Se (F) *** ** *** NS

Doses Se (D) *** NS *** ***

F � D *** NS *** ***

LSD 0.14 8.01 20.9 0.45

APX, ascorbate peroxidase; CAT, catalase; FW, fresh weight; GSH, glutathione; GSH-Px, glutathione peroxidase; LSD, least significant differences;

SOD, superoxide dismutase.aData are means � SE (n = 9). Levels of significance are represented by

*P < 0.05, **P < 0.01, ***P < 0.001 and NS, not significant.

0

100

200

300

400

500

600

700

800

0 5 10 20 40 60 80 120

µmol L-1

µg

Asc

orb

ic a

cid

g-1

FW

Se(VI) LSD0.05 31.20 Se(IV) LSD0.05 69.50

Figure 2 Total ascorbate concentration in lettuce plants subjected to different application rates and forms of Se. Data are means � SE (n = 9).

Selenium and antioxidant system in lettuce plants J.J. Rıos et al.

112 Ann Appl Biol 154 (2009) 107–116 ª 2008 The Authors

Journal compilation ª 2008 Association of Applied Biologists

form and rate of Se application (Table 1), generally pre-

senting greater growth when selenate was applied

instead of selenite (Table 1). In general, the results indi-

cate that selenate is less toxic than selenite because the

plants tolerated and responded positively to selenate that

even enhanced growth up to a rate of 20 lM for the

form selenate, while this effect for selenite occurred only

to a rate of 5 lM (Table 1).

Se-augmented LOX activity and foliar concentration of

MDA and selenite application induced higher values for

these parameters than did selenate (Table 1). MDA results

are similar to those found by Hartikainen et al. (2000),

who reported a dual role of Se in plant tissues: at low

concentration, Se acted as an antioxidant by diminishing

the lipid peroxidation, whereas at high concentration Se

acted as a pro-oxidant by increasing the accumulation of

thiobarbituric acid reactive substances. A similar behav-

iour was observed by Xue et al. (2001). However, our

data also coincide with works such as those of Lee &

Park (1998), which indicate that a selenite application

leads to a greater content in MDA than does a selenate

application. Thus, the data related to toxicity indicators

coincided with the decline in shoot biomass (Table 1),

agreeing with the finding in our experiment that Se

applied at high rates (80–120 lM) constitutes the most

phytotoxic treatment of our experiment, selenite being

the most phytotoxic Se form in our work.

One of the causes of lipid peroxidation is H2O2 accu-

mulation, which, like other ROS, attacks lipid mem-

branes, thereby increasing the quantity of MDA and

LOX activity (Djanaguiraman et al., 2005). Table 1 shows

an increase in the H2O2 concentration as the Se applica-

tion rate increases, its concentration being again higher

when the form used was selenite. This may explain why

selenite is more phytotoxic than selenate (Table 1).

For protection against ROS, plants contain antioxidant

enzymes such as SOD, CAT, APX and GSH-Px, as well as

a wide array of non-enzymatic antioxidants (Mittler,

2002; Blokhina et al., 2003). SOD is the enzyme in

charge of eliminating superoxide radicals and trans-

forming them into H2O2 (Bowler et al., 1994). In general,

the application of selenite to the culture medium boos-

ted SOD activity with respect to control, mainly from

20 lM (Table 2), suggesting greater oxidative stress in

these plants in comparison to plants treated with sele-

nate where the induction of SOD activity increased with

respect to control from 80 lM (Table 2). Our results are

similar to those reported by Hartikainen et al. (2000) and

Djanaguiraman et al. (2005), as these authors observed

greater SOD activity in ryegrass and soybean plants

when Se was applied in the form of selenite, principally

at the phytotoxic rates.

Catalase is found primarily in peroxisomes, transform-

ing H2O2 into water, although its affinity for the sub-

strate is weaker than that of other antioxidant enzymes

(Arora et al., 2002). The results indicate that there were

no significant differences in its activity with respect to

the Se application rate (Table 2), but we do find that the

selenate application augmented this activity with respect

to the plants treated with selenite at concentrations

greater than or equal to 20 lM (Table 2). In part, these

data are similar to those reported by Kong et al. (2005),

who found no effect of the Se application rate on CAT in

plants subjected to saline stress. Our results lead us to

conclude that the Se rate does not influence CAT activ-

ity, but the Se form does, suggesting that under stress

conditions the application of selenate would be more

beneficial than selenite. Finally, these results indicate

that in the presence of Se, CAT activity would not be the

main enzyme that detoxifies H2O2.

0

50

100

150

200

250

300

350

0 5 10 20 40 60 80 120

µmol L-1

nm

ol G

SH

g-1

FW

Se(IV) LSD0.05 12.75 Se(VI) LSD0.05 16.71

Figure 3 Total glutathione concentration in lettuce plants subjected to different application rates and forms of Se. Data are means � SE (n = 9).

J.J. Rıos et al. Selenium and antioxidant system in lettuce plants

Ann Appl Biol 154 (2009) 107–116 ª 2008 The Authors

Journal compilation ª 2008 Association of Applied Biologists

113

The enzymes APX and GSH-Px are in charge of detoxi-

fication of H2O2 produced by SOD in the chloroplast

through transformation of superoxide ion during photo-

synthesis (Arora et al., 2002). In general, the activity of

APX was higher with selenate than with selenite (Table 2).

In short, the lower APX activity and the absence of sig-

nificant variations in the CAT activity with respect to

control in plants treated with selenite (Table 2) together

with the increased SOD activity in these plants (Table 2)

could explain the higher concentration of H2O2 and

enhanced lipid peroxidation and, therefore, of the

decline in shoot biomass (Table 1) found after applying

rates higher than 20 lM of selenite. By contrast, sele-

nate application up to 60 lM caused variations in the

SOD activity and also increased the APX and GSH-Px

activities (Table 2), prevented greater foliar concen-

trations of H2O2 and lipid peroxidation (Table 1) and

caused a significant reduction in shoot biomass with

respect to control (Table 1).

The results for GSH-Px coincide with several studies

made with Se in which an increase in this trace element

augmented its activity (Hartikainen et al, 2000; Xue et al.,

2001; Djanaguiraman et al. 2005; Cartes et al., 2005).

However, our results indicate that the GSH-Px activity did

not have an essential role in H2O2 detoxification, at least

in the plants treated with selenite; such an increase in this

enzyme activity (Table 2) did not diminish the foliar con-

centration of H2O2 (Table 1). Finally, the different behav-

iour found in our study in terms of the GSH-Px activity

could be defined as a Se-dependent protein for which

activity depends exclusively on the presence of Se.

Both theAPX and theGSH-Px activities required reduced

compounds for their functioning. These compounds are

ascorbate and GSH, respectively (Arora et al. 2002). Pre-

viously, Rıos et al. (2008) observed that Se application

induced the synthesis of antioxidant compounds such as

total phenols, flavonoids, phenylpropanoids and glyco-

sides in lettuce plants, this induction being higher with

the application of selenate. In our experiment, the greater

induction of the synthesis and accumulation of different

antioxidants, such as ascorbate and GSH by selenate

(Figs 2 and 3), could also be explained by the fact that the

plants treated with this form of Se had lower oxidative

stress than those treated with selenite.

The results of GSH are important in this work, as they

indicate the degree of S assimilation and therefore the pos-

sibility of Se toxicity. According to Anderson (1993), Se

metabolism follows the same pathway as S, due to the

chemical similarity of the two molecules. Most enzymes

involved in S metabolism can catalyse the analogue reac-

tion with the corresponding Se substrates, forming Se ana-

logues of S compounds that are substrates for S enzymes

or replacing S in amino acids and thereby disrupting the

protein catalytic activity causing Se toxicity in plants.

Therefore, the greater formation of GSH in plants treated

with selenate (Fig. 3), together with a greater concentra-

tion in these plants of total S and total SH (data not

shown), could also explain why the application of this Se

form proved less phytotoxic than the application of sele-

nite. Possibly the highest incorporation of Se to amino

acids, especially Se-cysteine in plants treated with selenite

could provoke an alteration in the transport of e2 in pho-

tosynthesis, in consideration of the role of cysteine in this

process (Leustek et al., 2000), which would boost ROS

production in these plants. On the contrary, the lesser for-

mation of Se-cysteine in plants treated with selenate

together with a greater induction of the antioxidant sys-

tem could explain the lower ROS production and there-

fore less reduction of biomass production with respect to

plants treated with selenite.

In conclusion, we can indicate that the effect of Se on

the formation and detoxification of H2O2 in our work

depends largely on the form in which this trace element

is applied to the culture medium. Thus, the application

rate of selenite greater than 10 lM provoked a decline

in shoot biomass, associated with an increase in SOD

activity and a decrease in the enzymes in charge of H2O2

detoxification, mainly APX, which provoked a rise in

lipid peroxidation. On the contrary, in our work, the ap-

plication of selenate to 80 lM did not mean an increase

in the generation and accumulation of H2O2. Further-

more, the antioxidant system (CAT, APX, GSH-Px activi-

ties and total ascorbate and GSH concentrations) was

significantly improved with respect to control, which

could explain the beneficial effect of selenate found in

plants subjected to diverse abiotic stress. Finally, our re-

sults are novel and could define the application of sele-

nate at low application rates as a treatment to prevent

the induction of the antioxidant system in plants, help-

ing to improve the resistance responses to stress. How-

ever, it would be necessary to confirm these results in

future with studies that focus on the effect of Se during

the application time and the response of the different

isoenzymes that make up the antioxidant system in

plants.

Acknowledgements

This work was supported by PAI (Plan Andaluz de Inves-

tigacion, AGR-161).

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