Vol.2, No.2, 78-85 (2011)
doi:10.4236/as.2011.2 2012
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
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:
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
Copyright © 2011 SciRes. Openly accessible at http://www.scirp. org/journal/AS/
79
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-
2
4
SeO 
2
4
O
2
4
SeO 
Se

D. M. Ribeiro et al. / Agricultural Sciences 2 (2011) 78-85
Copyright © 2011 SciRes. http://www.scirp.org/journal/AS/
80
Root length (cm)
Al
Al+Se
Al+NAC
0
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
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.
2
SeO 
res 1 and 3).
The above results were completely different when
4
2
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
2
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-
2
SeO 
2
SeO 
2
SeO 
2
SeO 

, but without any effect on seedling
4
growth (as compared to Se-treated seedlings alone).
Openly accessible at

D. M. Ribeiro et al. / Agricultural Sciences 2 (2011) 78-85
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
81
Root length (cm)
- Se
+ Se
0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
MV (M)
Hypocotyl length (cm)
010
-7
10
-6
10
-5
10
-4
0
1.6
1.8
2.0
2.2
2.4
2.6
2.8
H
2
O
2
(M)
010
-7
10
-6
10
-5
10
-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).
2
4
SeO 
2
4
SeO 
2
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-
2
4
SeO 
2
4
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
4
SeO

effects on the control of seedling
growth, seedlings were treated with Al or Al plus 2
4
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
4
Se
2
4
SeO 
O

, respectively (not shown). In
keeping with those responses, Al toxicity effect was
– Se
0 10
–
7
10
–
6
10
–
5
10
–
4
0 10
–
7
10
–
6
10
–
5
10
–
4

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82
Root length (cm)
0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Hypocotyl length (c m)
0
1.6
1.8
2.0
2.2
2.4
2.6
2.8
Total seedling length (cm)
0
1
2
3
4
5
6
7
Ethylene (pmol seedling
-1
)
0
20
40
60
80
100
120
140
160
180
Control AVG NAC Se Se
+
AVG
Se
+
NAC
aaa
bb
a
B
C
B
A
C
A
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
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83
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.
2
4
SeO 
Se 2
4
O
2
4
SeO 
2
4
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.
2
4
SeO 
O
Se
2
4
SeO 
2
4
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
4
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
2
4
SeO 
2
4
SeO 
2
4
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.
2
4
SeO 
2
4

SeO
2
4
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
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84
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.
2
4
SeO 
2
4
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.
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