A dual role of selenium in the growth control of seedlings of Stylosanthes humilis

doi:10.4236/as.2011.22012

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Vol.2, No.2, 78-85 (2011)

doi:10.4236/as.2011.2 2012

Agricultural Sciences

A dual role of selenium in the growth control of

seedlings of Stylosanthes humilis

Dimas Mendes Ribeiro, Ana Maria Mapeli, Werner Camargos Antunes, Raimundo Santos

Barros*

Depto de Biologia Vegetal, Universidade Federal de Viçosa, Viçosa, Brazil; *Corresponding Author: [email protected]

Received 22 December 2010; revised 12 January 2011; accepted 22 February 2011.

ABSTRACT

The growth of seedlings of Townsville sytlo

(Stylosanthes humilis H.B.K.) is inhibited by

aluminium (Al) ions, their elongation being re-

covered with sodium selenate at 1.0 µM. Methyl

viologen and hydrogen peroxide, reactive

oxygen species (ROS)-generating compounds,

also inhibited seedling elongation and again

growth was relieved by selenate. Selenate, thus,

seemed to be operating as a ROS quencher,

since N-acetylcysteine (NAC), an antioxidant

compound, also stimulated largely the growth

of Al-inhibited seedlings. At a higher concentra-

tion (0.1 mM), how ever, selenate inhibited seed-

ling growth and elongation was recovered by

NAC. Ethylene production by selenate plus

NAC-treated seedlings was very higher and thus

the gaseous hormone was not responsible for

the seedling growth inhibition caused by sele-

nate. Hence, it seems that at high levels sele-

nate operates as a ROS-generating compound

whose effects were counteracted by NAC. It can

be deduced that, at low concentration, selenate

behaves as a ROS quencher an d at high level as

a ROS-promoting species.

Keyw ords: Aluminium; Ethylene; Growth Inhibition;

Reactive Oxygen Species; Selenate; Townsville

Stylo

1. INTRODUCTION

Plant growth is greatly affected by several environ-

mental stresses such as drought, extreme temperatures

and heavy metals. On acidic soils Al toxicity has been

recognized as a major limiting factor of plant productiv-

ity [1]. Plants can respond and adapt to Al stress by al-

tering their cellular metabolism and invoking various

defense mechanisms [2]. Usually Al toxicity induces

accumulation of reactive oxygen species (ROS), that

have been established as key signalling molecules con-

trolling a diverse range of physiological functions [3,4].

However, at high concentrations and in certain situations,

ROS may be toxic [5].

Since the effects of reactive oxygen molecules at cel-

lular level is mediated by their production and removal

via antioxidant activity [6], the use of free radical

quenchers may help to identify the role of ROS in plant

systems. Selenium (Se) is interesting in this matter be-

cause in response to oxidative stresses, Se compounds at

low concentrations, perform a protective function by

scavenging free radicals [7,8]. On the other hand, excess

Se can cause damage to plants, likely by triggering ROS

generation [9,10]. This antagonistic property makes Se

unique in studies dealing with systems requiring ROS to

elicit a physiological response.

Toxicity caused by Se compounds is an ill-understood

phenomenon [11]. There are indications that at high lev-

els Se can indiscriminately replace S in certain amino-

acids that are incorporated into proteins [11,12]. The

formation of Se-aminoacids, in turn, is supposed to en-

hance ethylene production [13], which can cause dam-

ages to plant growth. Some biochemical and physiologi-

cal studies were conducted with Se-compounds in plant

systems [8,14,15], but no physiological co-action be-

tween Se and Al has been stablished. In this work the

effects of Se at low concentration added to the growth

medium as a protector against Al toxicity were investi-

gated. Furthermore, the mode of action of Se, at high

concentration, on seedling growth of Townsville stylo

was also examined. The experiments were performed

with seedlings of Townsville stylo, an annual forage

legume cultivated in tropical pastures [16]. The species

has been considered as a potential contributor for pasture

improvement in tropical zones due to its high-quality

forage for livestock, high seed production and wide

adaptability to low fertility soils [17].

D. M. Ribeiro et al. / Agricultural Sciences 2 (2011) 78-85

2. MATERIALS AND METHODS

2.1. Plant Material and General Conditions

Seeds of Townsville stylo (Stylosanthes humilis H.B.K.)

were obtained from plants cultivated in 3.5 L plastic pots

in a greenhouse in Viçosa (20o45’ S, 42o15’ W), Minas

Gerais, Brazil and kept in the laboratory under dry con-

ditions. Non-dormant seeds were freed from their husks,

scarified with fine sandpaper (no 150), sterilized with 0.5

% NaOCl for 10 min, and thoroughly washed with dis-

tilled water. Seeds were taken to 15 cm diameter Petri

dishes with two layers of Whatman nº 1 filter paper and

16 ml of distilled water (pH 7.0). This assembly was

placed in the dark in a day/night growth chamber (Forma

Scientific Inc, Ohio, USA), at 30oC, for 18 h. Afterwards,

germinated seeds with a protruded radicle about 3 mm

long were transferred to 9 cm diameter Petri dishes with

two layers of filter paper, and incubated with 10 ml test

solutions. Solutions were prepared by dissolving chemi-

cals in 0.5 mM CaCl2 solution, pH 4.0, a condition that

prevents proton toxicity and leads to separation of the

effects of proton toxicity from the effects of Al toxicity

[18]. After 24 h exposure period, root and hypocotyl

lengths of the seedlings were determined.

2.2. Se Effects on Growth

Sodium selenate (Na2SeO4) was chosen as to repre-

sent the several soluble Se compounds whose effects on

dormancy breakage of Townsville stylo seeds are all

identical [14]. The effects of Na2SeO4, at low concentra-

tion (1 µM), on Al-treated seedling were examined by

providing the compound to seedlings in the solutions of

AlCl3 (1.0, 1.5 and 2 mM). In order to assess for a causal

association between Al-induced ROS production and

growth inhibition, seedlings were also exposed to com-

bined solutions of AlCl3 plus N-acetylcysteine (NAC, 1

mM), a free-radical quenching compound. Seedlings were

also exposed to solution of methyl viologen (MV, 10–7 -

10–4 M) and H2O2 (10–7 - 10–4 M), ROS-inducing sub-

stances, alone or to each one combined with Na2SeO4.

To investigate the effects of the exposure order seeds

were treated witch a combined solution of AlCl3 plus

Na2SeO4, AlCl3 or Na2SeO4 for the first 6 h. AlCl3 solu-

tions was then replaced by Na2SeO4 and AlCl3; Na2SeO4

solutions by AlCl3; seeds were kept in the new media for

18 h.

In order to search for the effects of Na2SeO4 at high

concentration (0.1 mM) on seedling growth and ethylene

production, Se-treated seedlings were also provided with

2-aminoethoxyvinylglycine (AVG, 10 µM) solution, an

inhibitor of ethylene biosynthesis. A putative relation-

ship between high Na2SeO4 concentration-induced ROS

generation and inhibition of seedling growth was also

searched for with the employment of NAC (1 mM).

2.3. Root Cell Viability

Cell viability was assessed by staining root tip frag-

ments with fluorescein diacetate (FDA, 10 μM) and

propidium iodide (PI, 2.0 μM), according to [19]. After

treatment with test solutions, seedlings were washed

with distilled water (pH 7.0) and roots tips were stained

for 5 min at room temperature with FDA and PI. The

viability of cells was observed under a fluorescent mi-

croscope (BH2, Olympus, Japan).

2.4. Ethylene Measurement

For ethylene quantification Erlenmeyer flasks (50 ml)

containing 10 seedlings imbibed in 3 ml test-solutions

were stoppered with rubber serum caps and kept in the

growth chamber, under the conditions previously des-

cribed. Air samples (1 ml) were taken from the flask

headspace and injected in a gas chromatograph (Hewlett

Packard 5890, Series II), equipped with a stainless-steel

column (1.0 m × 6.0 mm) packed with Porapak-N 80-

100 mesh. Ethylene quantitation was conducted under

the following conditions: nitrogen carrier gas and hydr-

ogen fluxes were 30 ml·min–1; air flux was 320 ml·min–1.

Column, injector and detector temperatures were 60, 110

and 150˚C, respectively. Ethylene peaks were registered

by a peak simple software (Peak Simple, Version 3.92)

coupled to the chromatograph, and quantified by com-

parison with authentic ethylene standards.

2.5. Statistical Analysis

The experiments followed a completely randomized

design, with 10 replications per treatment. Experimental

units consisted of a Petri dish or an Erlenmeyer flask

with 10 seedlings. The Tukey test at 5% was applied to

detect differences amongst means.

3. RESULTS

Aluminium inhibited root and hypocotyl growth of

Townsville stylo seedlings in a dose-dependent manner

(Figure 1). Inhibition of growth of Al-treated seedling

was alleviated by Na2SeO4, at the low concentration em-

ployed. Growth recovery by was about 95%,

81% and 66% in roots inhibited with 1.0, 1.5 and 2.0 mM

Al respectively. On the other hand, completely

counteracted the growth inhibition of hypocotyl caused

by Al. Selenate seemed to be operating through the

quenching of ROS since NAC, an antioxidant compound,

similarly recovered partially (roots) or completely (hy-

pocotyls) the growth of Al-inhibited seedlings (Figure 1).

Whether or not Al and were supplied to-

SeO 

O

SeO 

D. M. Ribeiro et al. / Agricultural Sciences 2 (2011) 78-85

Root length (cm)

Al+Se

Al+NAC

1.0

1.5

2.0

2.5

3.0

3.5

4.0

AlCl3 (mM)

Hypocotyl length (cm)

00.5 1.0 1.5 2.0

1.6

1.8

2.0

2.2

2.4

2.6

2.8

(d)

(c)

(b)

(a)

Figure 1. Se at low concentration alleviates AlCl3-in-

duced inhibition of seedling growth. AlCl3 was provided

to uniform seedlings in 0.5 mM CaCl2 soluton pH 4,0 or

combined with sodium selenate (1 µM) and N-acetyl

cisteine (NAC 1 mM). Means of 100 seedlings ± stan-

dard errors.

Figure 2. Protective effect of Se against Al-induced damages

in root tips of Townsville stylo seedlings. (a) Control; (b) 2

mM AlCl3; (c) 1 µM sodium selenate; and (d) AlCl3 plus so-

dium selenate. Healthy cells exhibit green fluorescence due to

fluorescein diacetate. Propidium iodide produces a red fluo-

rescence of nuclei in damaged cells. Photos are representative

of 5 replicates per treatment.

gether or separately, one of them anteceding or follow-

ing the supply of the other, their effects on seedling

growth were very similar (Table 1). Vital staining also

revealed differences in the response of seedlings treated

with Al plus 4 at low concentration (Figure 2).

Aluminium caused considerable damage to root cells of

Townsville stylo seedling. Selenate, on the other hand,

caused substantial reduction in the Al damages to roots.

SeO 

res 1 and 3).

The above results were completely different when

SeO



was used in a high concentration (0.1 mM), two

orders of magnitude larger than the one employed to

counteract the Al effects. At 0.1 mM, 4

SeO



inhibited

root and hypocotyl elongation by 48 and 21%, respec-

tively (Figure 4). As occurred with aluminium roots

were shown to be much more sensitive to high Se level

than hypocotyls. Selenate caused an increase in ethylene

emanation by seedlings by about 89% (as compared to

the control). The inhibitor of ethylene biosynthesis AVG

substantially decreased ethylene production by seedlings

treated with 2

SeO

That 4 seemed to be acting as an antioxidant

leading to growth alleviation or restoration of seedling

inhibited by Al was further demonstrated by treating

seedling with 4 and methyl viologen (MV) or

H2O2. Similarly to the Al effects, MV and H2O2 inhibited

root and hypocotyl elongation in a dose-dependent man-

ner (Figure 3). Furthermore, 4 restores (H2O2) or

alleviates (MV) the growth of inhibited roots; in hypo-

cotyls 4 completely overcame the inhibitory ef-

fects of both compounds. It is also observed that roots

were much more sensitive to Al than hypocotyls (Figu-

SeO 



, but without any effect on seedling

growth (as compared to Se-treated seedlings alone).

Openly accessible at

D. M. Ribeiro et al. / Agricultural Sciences 2 (2011) 78-85

Root length (cm)

- Se

+ Se

1.5

2.0

2.5

3.0

3.5

4.0

4.5

MV (M)

Hypocotyl length (cm)

010

-7

-6

-5

-4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

(M)

010

-7

-6

-5

-4

Figure 3. Effects of sodium selenate supplied with methyl viologen (MV, left) or H2O2 (right) solutions

pH 4.0 on seedling growth. Means of 100 seedlings ± standard errors.

Hence ethylene seemed not to be required for growth

inhibition of the -treated seedlings (Figure 4).

Selenate-induced inhibition of seedling growth was com-

pletely restored by NAC, but ethylene production by

-treated seedling was not decreased by the anti-

oxidant compound, which actually showed to be very

high (treatment plus NAC, Figure 4).

SeO 

4. DISCUSSION

Growth inhibition is a well-known response of plants

to toxic concentrations of Al ions [20]. The data de-

scribed herein demonstrate that , at low concen-

tration (1.0 µM), can alleviate partial or completely the

Al-induced inhibition of root and hypocotyl elongation,

respectively (Figure 1). Moreover, NAC, an antioxidant

compound [21], was also capable of overcoming the

inhibited state caused by Al. These results suggest that

the , at low concentration, may act as an antioxi-

dant (possibly as a ROS quencher) to counteract inhibit-

SeO 

tion of root and hypocotyl elongation of Townsville stylo

seedlings. In fact, Se can overcome oxidative damages

displaying a protective effect against stressing conditions

[8, 9]. Selenate addition restored hypocotyl growth to the

level of the control under any concentration of Al used.

However, root growth inhibition by Al (at 1.5 and 2.0

mM) was not recovered to the control level, indicating

that roots were more sensitive to a toxic Al exposure

than hypocotyls (see also Figure 3).

In order to get further insights into the relationships

between Al and 2

SeO



effects on the control of seedling

growth, seedlings were treated with Al or Al plus 2

SeO



(dissolved in 0.5 mM CaCl2, pH 4.0) for 24 h and after-

wards transferred to the medium without Al. Seedling

survival 10 days after transference was about 94%, 90%,

30%, and 80% if they were previously exposed to con-

trol (0.5 mM CaCl2, pH 4.0), 1 µM , 2 mM AlCl3

and AlCl3 plus 2

SeO 



, respectively (not shown). In

keeping with those responses, Al toxicity effect was

– Se

0 10

–

0 10

–

D. M. Ribeiro et al. / Agricultural Sciences 2 (2011) 78-85

Root length (cm)

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Hypocotyl length (c m)

1.6

1.8

2.0

2.2

2.4

2.6

2.8

Total seedling length (cm)

Ethylene (pmol seedling

-1

)

100

120

140

160

180

Control AVG NAC Se Se

AVG

NAC

aaa

Figure 4. Ethylene production is not associated with growth inhibition of high selenium con-

centration-treated seedlings. AVG (10 µM) and NAC (1 mM) were provided to seedlings in a

CaCl2 solution pH 4.0 alone or also combined with sodium selenate (0.1 mM). Means followed

by the same small letter (seedling growth), or followed by the same capital letter (ethylene) do

not differ significantly at 5% level. Data shown are means of 100 seedlings ± standard errors.

Ethylene (p mol·seedling–1)

D. M. Ribeiro et al. / Agricultural Sciences 2 (2011) 78-85

Ta bl e 1 . Effects of Na2SeO4 provided alone or in combination with AlCl3 on the growth of seedlings of Townsville stylo.

Sodium selenate (1 µM), AlCl3 (2 mM) and sodium selenate plus AlCl3 were provided to seedlings in 0.5 mM CaCl2 solu-

tion pH 4.0 for 6 h and then transferred to the next solutions (as indicated following the arrows) for 18 h. In each column

means do not differ significantly at 5% level, when followed by same letter. Means of 100 seedlings ± standard errors.

Treatment Root length (cm) Hypocotyl length (cm) Seedling length (cm)

Control → Control 3.8 ± 0.09 a 2.4 ± 0.04 a 6.2 ± 0.10 a

Se → Se 3.6 ± 0.08 a 2.5 ± 0.04 a 6.1 ± 0.10 a

AlCl3 → AlCl3 1.2 ± 0.07 c 1.8 ± 0.05 b 3.0 ± 0.09 c

AlCl3 → Control 1.4 ± 0.08 c 1.9 ± 0.05 b 3.3 ± 0.11 c

AlCl3 → Se 2.4 ± 0.10 b 2.5 ± 0.04 a 4.9 ± 0.10 b

Se → AlCl3 2.7 ± 0.12 b 2.3 ± 0.05 a 5.0 ± 0.13 b

AlCl3 + Se → AlCl3 + Se 2.4 ± 0.11 b 2.4 ± 0.06 a 4.7 ± 0.15 b

very high in seedling expose to Al 2 mM, as shown by

vital staining of root tips (Figure 2). Selenate substan-

tially reduced cell damages caused by Al, also diminish-

ing the Al inhibition of root elongation, evidecing a pro-

tective role of Se. The inhibition relief of Al-treated

seedling by was also observed had the seedlings

been exposed to before (Se → Al) or after (Al

→ Se) the Al supply (Table 1). Moreover, pre-treatment

of seedlings with (Se → Al) or with Al (Al →

Se) promoted a similar effect on root and hypocotyl

elongation as the combined treatment with Al plus

. It follows that in short term Se is capable of im-

pairing or repairing the damages caused by aluminium in

the tissues.

SeO 

Se 2

O

SeO 

A probable role of , at low concentration, as an

antioxidant agent to alleviate the Al-induced inhibition

of seedling growth was also examined with the employ-

ment of MV and H2O2. MV, which generates singlet

oxygen (2) directly and OH• radicals as secondary

activated oxygen species [22,23], and H2O2 constitute

important tools for investigating the effects of activated

oxygen species in biological systems. Similarly to the

treatment with Al, both MV and H2O2 reduced seedling

growth and their inhibitory effect was substantially re-

versed by , at the low concentration employed

(Figure 3). These results suggest that the diminished

action of oxygen species could explain the protective

role of in Al-stressed plants.

SeO 

O

SeO 

O

Openly accessible at

The results point out to Se as exerting a dual effect on

seedling growth process: at low concentration, it could

act as an antioxidant enhancing growth of Al-inhibited

seedlings, whereas at higher concentration it could act as

a growth-inhibiting agent [9,24]. In fact, selenomethion-

ine at high concentration inhibited seedling growth, as

shown in [25], and several Se-soluble compounds elic-

ited ethylene production by seeds and seedlings of

Townsville stylo [14]. It is known that selenate at high

levels, upregulates the genes coding for 1-aminocyclo-

propane-1-carboxylic acid (ACC) synthase and ACC

oxidase, the two last enzymes in the pathway to ethylene

biosynthesis [26]. Root elongation is also inhibited by

ethylene [27] and thus it is likely that 2

SeO



, at high

concentration, inhibits seedling growth through eliciting

ethylene biosynthesis (Figure 4). However, AVG inhibited

ethylene production of -treated seedlings to a great

extent, without any effect on seedling growth in compari-

son to seedlings treated with solely (Figure 4).

These findings suggest that other mechanisms of action of

SeO 

SeO



at high concentrations, might be operative, which

was supported with the use of NAC. When NAC was

supplied to seedling together with , seedling

growth was increased to the level of the control and eth-

ylene production stimulated by was not inhib-

ited at all by NAC (Figure 4). Together these data pro-

vide evidence supporting that -induced inhibition

of seedling growth was likely associated with the action

of ROS and not with ethylene production.

SeO 



SeO

SeO 

The results obtained on radicle growth of Towsville

stylo seedlings are in contrast to those of bean, another

legume [28], and Arabdopsis thaliana, a Brassicaceae

[27]. In these species radicle growth is inhibited by the

large amounts of ethylene induced by Al. In Arabidopsis

the effects of ethylene were much reduced in the mutants

etr-1 and ein-2, defective in ethylene signalling or with

the use of AVG, Co2+ and with Ag+, inhibitors of ethyl-

ene biosynthesis and action. By also employing the

mutants aux1-7 and pin2, defective in auxin polar trans-

port, and using naphthylphthalamic acid, winch disrupts

the auxin polar transport, the inhibitory effects of Al in

radicle growth were also greatly diminished or no longer

observed. It was concluded that ethylene constitutes a

signal which alters auxin distribution in roots by dis-

rupting AU X1 and PI N 2-mediated auxin polar transport,

causing an arrest in root elongation [29]. There remains,

however, the possibility of a direct action of Al3+ in

auxin distribution, thus bypassing the ethylene require-

ment, [29]. In this context, it can be conclused that the

causes for inhibition of root growth are species-specific.

D. M. Ribeiro et al. / Agricultural Sciences 2 (2011) 78-85

In summary, the action of on seedling growth

of Townsville stylo seedling was shown to depend on its

concentration. At low concentration, it promotes the re-

lease of the Al growth inhibition, seeming to work as a

scavenger of free radicals. At high concentrations, inhi-

bition of seedling growth is not associated with a

-dependent ethylene biosynthesis, but seems as-

sociated to the ROS generation.

SeO 

5. ACKNOWLEDGEMENTS

Thanks are due to FAPEMIG (Foundation for Research Support of

Minas Gerais State) for the post-doctoral fellowship awarded to D.M.R.

and for the financial support during the conduct of this research.

REFERENCES

[1] Delhaize, E. and Ryan, P.R. (1995) Aluminum toxicity

and tolerance in plants. Plant Ph ys iol ogy, 107, 315- 321.

[2] Kochian, L.V., Hoekenga, O.A. and Pineros, M.A (2004)

How do crop plants tolerate acid soils? Mechanisms of

aluminium tolerance and phosphorous efficiency. Annual

Review of Plant Biol og y, 55, 459-493.

doi:10.1146/annurev.arplant.55.031903.141655

[3] Neill, S.J., Desikan, R. and Hancock, J.T. (2002) Hydro-

gen peroxide signalling. Current Opinion in Plant Biol-

ogy, 5, 388-395. doi:10.1016/S1369-5266(02)00282-0

[4] Oracz, K., Bouteau, H,E.M., Farrant, J. M., Cooper, K.,

Belghazi, M., Job, C. et al. (2007) ROS production and

protein oxidation as a novel mechanism for seed dor-

mancy alleviation. The Plant Journal, 50, 452-465.

d oi :1 0.1111 /j .13 65-313X.2007.03063.x

[5] Bailey-Serres, J. and Mittler, R.M. (2006) The roles of

reactive oxygen species in plant cells. Plant Physiology,

141, 331. doi:10.1104/pp.104.900191

[6] Carol, R. and Dolan, L. (2006) The role of reactive oxy-

gen species in cell growth: Lessons from root hairs.

Journal of Experimental Botany, 57, 1829-1834.

doi:10.1093/jxb/erj201

[7] Seppänen, M., Turakainen, M. and Hartikainen, H. (2003)

Selenium effects on oxidative stress in potato. Plant Sci-

ence, 165, 311-319.

doi:10.1016/S0168-9452(03)00085-2

[8] Filek, M., Keskinen, R., Hartikainen, H., Szarejko, I.,

Janiak, A., Miszalski, Z. et al. (2008) The protective role

of selenium in rape seedlings subjected to cadmium

stress. Journal of Plant Physiology, 165, 833-844.

doi:10.1016/j.jplph.2007.06.006

[9] Hartikainen, H., Xue, T. and Piironen, V. (2000) Sele-

nium as an antioxidant and pro-oxidant in ryegrass. Plant

and Soil, 225, 193-200.

doi:10.1023/A:1026512921026

[10] Mora, M., Pinilla, L., Rosas, A. and Cartes, P. (2008)

Selenium uptake and its influence on the antioxidative

system of white clover as affected by lime and phospho-

rus fertilization. Plant and Soil, 303, 139-149.

doi:10.1007/s11104-007-9494-z

[11] Brown, T.A. and Shrift, A. (1982) Selenium: Toxicity and

tolerance in higher plants. Biological Review, 57, 59-84.

d oi :1 0.1111 /j .14 69-185X.1982.tb00364.x

[12] Eustice, D.C., Kull, F.J. and Shrift, A. (1981) Selenium

toxicity: Aminoacylation and peptide bond formation

with selenomethionine. Plant Physiology, 67, 1054- 1058.

doi:10.1104/pp.67.5.1054

[13] Konze, J.R., Schilling, N. and Kende, H. (1978) En-

hancement of ethylene formation by selenoamino acids.

Plant Physiology, 62, 397-401.

doi:10.1104/pp.62.3.397

[14] Pinheiro, F.J.A., Barros, R.S., Coelho, T.G. and Souza, B.

M.L. (2008) Breaking dormancy of Stylosanthes humilis

seeds with selenium compounds. Seed Science Research,

18, 47-53. doi:10.1017/S0960258508890253

[15] Feng, R., Wei, C., Tu, S. and Sun, X. (2009) Interactive

effects of selenium and arsenic on their uptake by Pteris

vittata L. under hydroponic conditions. Environmental

and Experimental Botany, 65, 363-368.

doi:10.1016/j.envexpbot.2008.11.013

[16] Williams, R.J., Reid, R., Schultze-Kraft, R., Souza Costa,

N.M. and Thomas, B.D. (1984) Natural distribution of

Stylosanthes. In: Stace, H.M. and Edye, L.A. Eds., The

Biology and Agronomy of Stylosanthes, Academic Press,

Sydney, 73-110.

[17] Gardner, C.J. (1984) The Dynamics of Stylosanthes Pas-

tures. In: Stace, H.M. and Edye, L.A. Eds., The Biology

and Agronomy of Stylosanthes, Academic Press, Sydney,

333-357.

[18] Rangel, A.F., Mobin, M., Rao, I.M. and Horst, W.J. (2005)

Proton toxicity interferes with the screening of common

bean (Phaseolus vulgaris L.) genotypes for aluminium

resistance in nutrient solution. Journal of Plant Nutrition

and Soil Science, 168, 607-616.

doi:10.1002/jpln.200520509

[19] Jones, K.H. and Senft, J.A. (1985) An improved method

to determine cell viability by simultaneous staining with

fluorescein diacetate-propidium iodide. Journal of His-

tochemistry and Cytochemistry, 33, 77-79.

doi:10.1177/33.1.2578146

[20] Jones, D.L., Blancaflor, E.B., Kochian, L.V. and Gilroy,

S. (2008) Spatial coordination of aluminium uptake,

production of reactive oxygen species, callose production

and wall rigidification in maize root plant. Plant Cell and

Environment, 29, 1309-1318.

d oi :1 0.1111 /j .13 65-3040.2006.01509.x

[21] Lobréaux, S., Thoiron, S. and Briat, J.-F. (1995) Induc-

tion of ferritin synthesis in maize leaves by an

iron-mediated oxidative stress. The Plant Journal, 8,

443-449. doi:10.1046/j.1365-313X.1995.08030443.x

[22] Babbs, C.F., Phan, J.A. and Coolbaugh, R.C. (1989) Le-

thal hydrogen radical production in paraquat-treated

plants, Plant Physiogy, 90, 1267-1270.

doi:10.1104/pp.90.4.1267

[23] Foyer, C.H., Lelandais, M. and Kunert, K.J. (1994)

Photooxidative stress in plants. Physiologia Plantarum,

92, 696-717. doi: 10.1111 /j .1399-3054.1994.tb03042.x

[24] Xue, T., Hartikainen, H. and Piironen, V. (2001) Anti-

oxidative and growth-promoting effect of selenium on

senescing lettuce. Plant and Soil, 237, 55-61.

doi:10.1023/A:1013369804867

[25] Barros, R.S. and Freitas, A.W.P. (2001) Selenomethion-

ine as a dormancy-breaking agent in seeds of Stylosan-

thes humilis. Acta Physiologicae Plantarum, 23, 279-284.

doi:10.1007/s11738-001-0034-9

D. M. Ribeiro et al. / Agricultural Sciences 2 (2011) 78-85

[26] Hoewyk, D.V., Takahashi, H., Inoue, E., Hess, A., Tam-

aski, M. and Pilon-Smits, E.A.H. (2008) Transcriptome

analyses give insights into selenium-stress responses and

selenium tolerance mechanisms in Arabidopsis. Physi-

ologia Plantaru m, 132, 236-256.

doi:10.1093/pcp/pcm077

[27] Sun, P., Tian, Q.-Y., Zhao, M.G., Dai, X.Y., Huang, J.H.,

Li, L.H. et al., (2007) Aluminum-induced ethylene pro-

duction is associated with inhibition of root elongation in

lotus japonicus L. Plant and Cell Physiology, 48,

1229-1235.

[28] Massot, N., Nicander, B., Barcelo, J., Poschenrieder, C.H.

and Tillberg, E. (2002) A rapid increase in cytokinin lev-

els of root growth in bean seedlings (Phaseolus vulgaris

L). Plant Growth Regulation, 37, 105-112.

doi:10.1023/A:1020511007138

[29] Sun, P., Tian, Q.-Y., Chan, J. and Zhang, W.-H. (2010)

Aluminium-induced inhibition of root elongation in

Arabidopsis is mediated by ethylene and auxin. Journal

of Experimental Botany, 61, 347-356.