ORIGINAL ARTICLE

Mitigating effects of acetylcholine supply on soybean seed
germination under osmotic stress

Inaê Braga1 • Maria Dolores Pissolato2 • Gustavo Maia Souza3

Received: 18 July 2016 / Accepted: 6 February 2017 / Published online: 23 February 2017

� Botanical Society of Sao Paulo 2017

Abstract Acetylcholine (Ach) is a common neurotrans-

mitter in animals, also synthesized in plants, which can

have an influence on plant response to stress, also acting as

a signaling molecule between root and shoot. The objective

of this study was to analyze the possible mitigating effects

of exogenous application of Ach on soybean germination

under different levels of osmotic potential. The experi-

ments were conducted with soybean [Glycine max (L.)

Merrill] genotype Intacta. The seeds were first treated with

Ach solutions with the following concentrations: 0.0

(control); 0.5; 1. 0 and 2.0 mM. Then, the seeds were

subjected to two water potentials, -0.5 and -1.0 MPa,

reached by using mannitol solutions, for the induction of

osmotic stress, and a control condition with distilled water.

Thus, 12 treatments were established in a double factorial

4 9 3, with 4 levels of Ach and 3 osmotic potential

treatments (0.0, -0.5 and -1.0 MPa) with four replicates

per treatment. The results showed that the concentration of

1.0 mM Ach, without osmotic stress, presented higher

values for total dry mass of the seedlings compared to the

control treatment (without Ach supply). In the treatments

conducted to test the effectiveness of Ach on the mitigation

of severe osmotic stress effects (-1.0 MPa), results

showed that the concentration of 0.5 mM Ach showed

positive results for the following parameters; dry weight

of shoot, root dry weight, total dry mass, which were

significantly higher than treatment under 1.0 MPa.

Keywords Abiotic stress � Glycine max �
Neurotransmitter � Seedling growth

Introduction

Soybean [Glycine max (L.) Merrill] is originally from

China and belongs to the Fabaceae family (Sediyama et al.

2009). Soybean seeds contain about 40–45% protein and

18–20% oil in their composition (Islam et al. 2010). It is

one of the major commercialized crops of the world,

considered to be a major commodity, and the main pro-

ducers are the USA, Brazil and Argentina, which are also

the main exporters (Barzotto et al. 2012).

Seed quality can be affected by several factors during

the process of plant development under field conditions.

Factors such as inadequate temperatures, water deficit in

the soil, along with other environmental variables can slow

down the process of germination and seedling emergence,

causing the seeds to remain exposed to unfavorable soil

conditions for a longer time (Barzotto et al. 2012). Water

availability is a major limiting factor of germination and

early seedling development in field conditions, since it has

an important role in the activation of different metabolic

processes with a straightforward relation to germination of

seeds (Avila et al. 2007).

Drought is characterized as an abiotic stress factor

related to decreased water content in the soil, subjecting the

plants to water stress (Paiva and Oliveira 2006). Water

& Inaê Braga

inae_braga@yahoo.com.br

1 Programa de Pós-Graduação em Biologia Vegetal, Instituto

de Biociências, Universidade Estadual Paulista ‘‘Júlio de

Mesquita Filho’’ (UNESP), Rio Claro, SP, Brazil

2 Graduação em Ciências Biológicas Bacharelado,

Universidade do Oeste Paulista (UNOESTE), Rod. Raposo

Tavares, km 572, Presidente Prudente, SP CEP 19067-175,

Brazil

3 Departamento de Botânica, Universidade Federal de Pelotas

– UFPel, Campus Universitário, S/N, Cx. Postal 345, Pelotas,

RS, Brazil

123

Braz. J. Bot (2017) 40(3):617–624

DOI 10.1007/s40415-017-0367-2

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stress is associated with reduced water availability and

cellular dehydration (Maraghni et al. 2010). As the soil

water content or the substrate decreases, initially it appears

to reduce the germination rate and, consequently, tighter

restrictions can operate upon seedling development (Mar-

cos Filho 2005). For each species, there is an ideal amount

of soil water potential, below which there is limited or even

a suppression of germination (Maraghni et al. 2010).

In order to perform studies on the of response of plants

subjected to water stress under laboratory conditions,

aqueous substances with different levels of osmotic

potential are used (Machado et al. 2001). Several sub-

stances are used in these studies to induce osmotic stress,

such as PEG (polyethylene glycol), which, due to its

molecular weight, cannot enter cells, and mannitol, which

is a chemically inert molecule and non-toxic for plants

(Avila et al. 2007).

Seeds subjected to water stress show a reduction in both

growth speed and germination rate due to the decreased

enzyme activity, inducing lower meristem development

(Popinigis 1985; Avila et al. 2007). However, there are

substances present in the seeds that can confer protection

when subjected to stress, such as gibberellic acid, abscisic

acid, ethylene and cellular compatible osmolytes (Mana-

valan et al. 2009; Balardin et al. 2011).

Recently, neurotransmitter molecules have also been

found in plants performing functions related to the reg-

ulation of development under stress (Xu and Hu 2014).

Among the most widespread neurotransmitters studied in

plants are c-aminobutyric acid (GABA) and acetyl-

choline (Ach) (Bouché and Fromm 2004), adrenaline,

noradrenaline, dopamine, serotonin and glutamate (Ros-

china 2001). These molecules have been related to the

activation of seed and pollen germination, regulation of

ion permeability, energy balance, morphogenesis,

movements of root, leaves and stomata, regulation of

electrical events and protection against stress (Roschina

2001).

Ach is a small neurotransmitter molecule widespread

throughout the nervous system of animals (Wessler and

Kirkpatrick 2008). It has also been found in organisms

without nervous system such as bacteria, algae, protozoa,

poriferans, fungi and plants (Horiuchi et al. 2003; Sagane

et al. 2005). In plant’s cells, Ach is synthesized in the

stems, leaves, nodes and roots in response to stress, par-

ticipating in the signaling processes between the root and

the shoot (Murch 2006). Choline and acetyl-coenzyme A

are responsible for Ach synthesis mediated by the enzyme

choline acetyltransferase (ChAT). Once the Ach biosyn-

thesis rate is correlated with the presence of their precur-

sors and associated enzymes, Ach can influence the process

of biosynthesis by regulating enzymes such as ChAT,

acetyl-CoA carboxylase, choline kinase, cholinesterase and

pseudocholinesterase (Wessler et al. 2001). The hydrolysis

of Ach and its metabolic derivatives, choline and acetate, is

made by the enzyme acetylcholinesterase (AChE) (Blusz-

tajn et al. 1987).

Studies have shown that osmotic stress causes a

decrease in the level of Ach in the roots and in the leaf

abaxial epidermis of Vicia faba L. even when the shoot was

hydrated, affecting stomatal movement. Thus, it is evident

the importance of Ach in signal transduction between shoot

and root, including in response to stress (Wang et al. 2003).

According to Wang et al. (2003), Ach may have a stimu-

latory effect on stomatal control. This control is due to the

presence of Ach receptors, called ‘‘ Ach binding sites,’’

found in the chloroplasts (Roschina 2001), in the mem-

brane vacuoles (Gong and Bisson 2002) and in the plasma

membrane of guard cells (Meng et al. 2001). In radish

germination (Raphanus sativus L.), grown with and with-

out addition of Ach, the growth and development of lateral

roots into seedlings was observed, but no effect on the main

stems and roots was found (Sugiyama and Tezuka 2011).

According to Daneluzzi (2012), the effects of Ach on

germination and plant growth changes from species to

species, calling for additional studies to elucidate its role in

plant development. Thus, the main objective of this study

was to analyze a possible mitigating effect of the exoge-

nous supply of Ach on soybean germination under different

levels of osmotic potential.

Materials and methods

The experiments were carried out with soybean [Glycine

max (L.) Merrill] genotype Intacta.

Seeds were washed with distilled water and treated with

systemic fungicide Vitavax (0.5 mL for 200 g of seed). For

germination were used 200 seeds per treatment, disposed in

three sheets of paper towel-type germitest (two sheets of

base and cover), wrapped in a transparent plastic bag.

Treatments with different concentrations of Ach were

equivalent to 2.5 times the weight of the dry substrate of

solutions with different Ach concentrations (0.0, 0.5, 1.0

and 2.0 mM) directly on the germitest sheets. Germination

was carried out under different osmotic conditions (-0.5

and -1.0 MPa) using two concentrations of mannitol (74.3

and 37.15 g L-1, respectively), and a control treatment

without mannitol, established using distilled water.

Therefore, the experiment was performed on a factorial

design 4 9 2 (four Ach concentrations and two osmotic

conditions) and four replicates.

The experiment was carried out in a Mangelsdorf-type

germination chamber (ELETROLAB, São Paulo, BR) with

a photoperiod of 12 h and temperature of 28 and 25 �C
day/night.

618 I. Braga et al.

123



Analyzed variables – The germination test followed

the Rules for Seed Analysis (Brasil 2009). Germination

(%G)—evaluations were performed at five (first count)

and eight (final count) days after seeding was regarded

as the number of seeds that produced normal seedlings,

that is, the emergence and development of the main

embryonic structures (Brasil 2009). Germination speed

index (GSI)—calculated according to Maguire (1962).

The first count (FC%)—the percentage data used nor-

mal seedlings on the fifth day after the germination

test.

Seedling vigor classification—the classification of

normal seedlings in three categories: strong normal

(SNS, vigorous), week normal (WNS, little vigorous) and

abnormal (problem in your structure or injury) (Naka-

gawa 1999; Brasil 2009). Dry matters of seedlings—to

test the growth of seedling, four replicates of 10 seeds

each were used. The seeds were germinated according to

the methodology for the test germination. The parameters

were evaluated as follows: length of root (LR), shoot

(LS) and total length ðLT ¼ LRþ LSÞ; dry mass of the

shoot (DMS), root (RMD) and dry matter total

ðDMT ¼ DMSþ RMDÞ, and by calculating the rela-

tionship between the dry mass of root and shoot (R/S).

The dry mass analysis was conducted after oven drying

at 60� until a constant mass was achieved (Nakagawa

1999).

Data analysis – The data have been subjected to anal-

ysis of variance (ANOVA, P = 0.05) and the mean of

values were compared via the Tukey test (P\ 0.05), using

the statistical software SISVAR (5.1, Federal University of

Lavras).

Additionally, the normalized variation index (NIV) was

calculated using the following equation as described by

Tattini et al. (2006):

NIVAch on control ¼ ðXAch � XcontrolÞ=ðXAch þ XcontrolÞ
NIVAch onmannitol ¼ ðXAch � XmannitolÞ=ðXAch þ XmannitolÞ

where XAch values are the concentrations of Ach (0.5,

1.0 and 2.0 mM) and Xcontrol is the treatment without

Ach supply. These calculations were performed sepa-

rately considering 1—treatments with different concen-

trations of Ach with distilled water (Fig. 1), and 2—

treatments under -1 MPa with different concentrations

of Ach, and Xmannitol is the treatment without Ach supply

(Fig. 2).

The NIV is a standardized index of phenotypic plastic-

ity, allowing for the visualization of the real effect of the

treatment via the analyzed parameters, taking positive or

negative values in relation to the control treatment, and the

mannitol treatment, with distilled water or under -1 MPa,

respectively.

Fig. 1 Normalized variation index of percentage of germinated

(%G), germination speed index (GSI), first count (%FC), strong

normal seedlings (SNS), weak normal seedlings (WNS), length shoot

(LS), length root (LR), length total (LT), dry matter of shoot (DMS),

root dry mass (RDM), total dry matter (DMT) and relationship

between root and shoot (R/S) analyzed during the germination process

[Glycine max (L.) Merrill] under Ach 0.0 MPa and osmotic condi-

tions. The dotted horizontal line shows the normalized values of the

treatment without the application of Ach

Fig. 2 Normalized variation index of percentage of germinated

(%G), germination speed index (GSI), first count (%FC), strong

normal seedlings (SNS), weak normal seedlings (WNS), length shoot

(LS), length root (LR), length total (LT), dry matter of shoot (DMS),

root dry mass (RDM), total dry matter (DMT) and relationship

between root and shoot (R/S) analyzed during the germination process

[Glycine max (L.) Merrill] under conditions to Ach and osmotic

-1.0 MPa (induced by mannitol). The dotted horizontal line shows

the normalized values of the treatment without the application of Ach

Mitigating effects of acetylcholine supply on soybean seed germination under osmotic stress 619

123



Results

The results of the exogenous supplying of Ach in the

treatments without osmotic stress indicated that the con-

centration of 2.0 mM showed positive effects on seedling

growth parameters such as LR, LT, DMS and RDM com-

pared to the control treatment (no Ach addition). As well as

the observations in the 2.0 mM concentration, the treat-

ments with 1.0 and 0.5 mM also induced a significant

increase in DMT (DMS, and RDM). However, there was a

decrease in the root and shoot relationship for all Ach

concentrations evaluated when compared to control. On the

other hand, no significant differences were observed for the

germination parameters evaluated (Table 1).

In the treatments under -1.0 MPa of water potential,

there was a significant reduction in all parameters evalu-

ated, compared to the treatments with distilled water. On

the other hand, there were no significant effects observed

on the germination and initial growth of seedlings under

water potential of -0.5 MPa (data not shown). Thus, these

results will not be considered later.

In the treatments performed to test the effectiveness of

Ach on mitigating the effects of severe osmotic stress

(-1.0 MPa), the results showed that the concentration of

0.5 mM exhibited positive results on SNS, DMS, RDM,

DMT, which were significantly higher than the -1.0 MPa

treatment without the addition of Ach. The results showed

that the concentration of 0.5 mM caused positive effects

on SNS, DMS, RMD, DMT, which were significantly

higher than the -1.0 MPa treatment without the addition

of Ach. The concentration of Ach 1.0 mM showed a

significant increase in the root–shoot relationship when

compared to all other concentrations, including the con-

trol, while the concentrations of 0.5 and 2.0 mM were

statistically similar, inducing an increase in the %G

(Table 1).

The NIVcontrol analysis with the different concentrations

of Ach without the osmotic agent (Fig. 1) showed no sig-

nificant differences among treatments in relation to ger-

mination traits. On the other hand, when taking into

account the growth parameters of the seedlings, the treat-

ment with 2.0 mM of Ach showed increased NIVcontrol in

the parameters of LR and LT (0.3612 and 0.1464, respec-

tively). A subtle decrease of -0.1973 was observed in the

LS values in the concentration of 0.5 mM. However, the

dry mass parameters in the concentration of 1.0 mM

showed an increase in the NIVcontrol values of DMS

(0.6154), RDM (0.3846) and DMT (0.5054). Moreover, a

decrease was observed in NIVcontrol for the R/S ratio for all

the Ach concentrations analyzed.

The results of NIVmannitol in seeds under osmotic stress

(Fig. 2) showed a positive increase in all parameters

analyzed; germination, growth and dry weight, in the three

concentrations of Ach (0.5, 1.0 and 2.0 mM) .

The concentration of 0.5 mM showed higher NIVmannitol

values for the parameters of LR (0.4127), LT (0.5066),

DMS (0.6667), RDM (0.8333) and DMT (0.7222). The

highest values of NIVmannitol were observed at R/S (0.6498)

in the treatment with 1.0 mM of Ach.

Discussion

In conditions without osmotic stress, the application of Ach

showed a positive trend on the development parameters of

the seedlings, but not on the germination traits. Many

studies have suggested the involvement of Ach in regu-

lating physiological processes in plants, especially in plant

growth and in the development processes controlled by

phytochrome (Tretyn et al. 1990). The Ach can induce

similar effects to red light in the regulation of some pho-

tomorphogenic phenomena (Jaffe 1970; Tretyn 1987;

Hartmann and Gupta 1989). Wild plants etiolated under red

light irradiation showed an increase in the endogenous

levels of Ach, and after application of a far-red light pulse,

the reversal of this effect was observed (Wiśniewska and

Tretyn 2003), suggesting that Ach must be related to the

etiolation of plants, such as was observed herein with the

application of 1 mM in the germinated seeds under

-1.0 MPa (Fig. 2). In this treatment, there was a greater

increase in the LS (NVI = 0.5547) in relation to the DMS

(NVI = 0.2000) compared with the treatment without the

addition of Ach.

In this study, it was observed that the application of Ach

in plants under osmotic stress -1 MPa (Table 1) promoted

an elongation of the roots of seedlings. This phenomenon is

dependent on the metabolic activity resultant in induction

or inhibition of enzyme activity regulators in plant growth

(Sugiyama and Tezuka 2011). Sugiyama and Tezuka

(2011) carried out experiments with the application of Ach

and observed the emergence and elongation of lateral roots

in Vigna sesquipedalis (L.) Fruwirth, Avena sativa L. and

Raphanus sativus (radish) grown in the dark. In particular,

the effect of Ach was remarkable on radish seedlings.

Different concentrations of Ach (0.1–100 nM), mainly

1 nM, induced higher growth, emergence and lateral root

elongation, compared with control plants (Sugiyama and

Tezuka 2011). According to Bamel et al. (2007), the for-

mation of adventitial roots and secondary roots in detached

leaves of tomatoes (Lycopersicon esculentum Mill.) grown

in vitro was promoted by concentrations between 10-3 and

10-7 M, wherein 10-5 M showed the best results.

Because rooting (in vivo or in vitro) is mainly affected

by auxin, the effectiveness of Ach in promoting root grown

620 I. Braga et al.

123



T
a
b
le

1
P
er
ce
n
ta
g
e
o
f
g
er
m
in
at
ed

(%
G
),
g
er
m
in
at
io
n
sp
ee
d
in
d
ex

(G
S
I)
,
fi
rs
t
co
u
n
t
(%

F
C
),
st
ro
n
g
n
o
rm

al
se
ed
li
n
g
s
(S
N
S
),
w
ea
k
n
o
rm

al
so
y
b
ea
n
se
ed
li
n
g
s
(W

N
S
),
le
n
g
th

sh
o
o
t
(L
S
),
le
n
g
th

ro
o
t
(L
R
),
le
n
g
th

to
ta
l
(L
T
),
d
ry

m
at
te
r
o
f
sh
o
o
t
(D

M
S
),
ro
o
t
d
ry

m
as
s
(R
D
M
),
to
ta
l
d
ry

m
at
te
r
(D

M
T
)
an
d
re
la
ti
o
n
sh
ip

b
et
w
ee
n
ro
o
t
an
d
sh
o
o
t
(R
/S
)
so
y
g
en
o
ty
p
e
In
ta
ct
a,
su
b
je
ct
ed

to
d
if
fe
re
n
t

co
n
ce
n
tr
at
io
n
s
o
f
ac
et
y
lc
h
o
li
n
e
an
d
d
if
fe
re
n
t
o
sm

o
ti
c
co
n
d
it
io
n
s

O
sm

ó
ti
co

(M
P
a)

C
o
n
tr
o
l
(0
.0
)

M
an
n
it
o
l
(-

1
.0
)

A
ch

(m
M
)

0
0
.5

1
2

0
0
.5

1
2

%
G

7
2
.5
0
A
a
±

1
.8
4

6
1
.0
0
A
ab

±
1
.7

6
4
.5
0
A
ab

±
0
.8
5

4
9
.5
0
A
b
±

3
.1
9

3
9
.5
0
B
b
±

1
.2
5

5
7
.0
0
A
a
±

2
.5

5
1
.0
0
B
ab

±
1
.5

5
9
.9
9
A
a
±

1
.4

G
V
I
(d
)

5
.2
2
A
a
±

0
.2
8

4
.2
7
A
ab

±
0
.2
7

4
.4
1
A
ab

±
0
.1
8

3
.4
5
A
b
±

0
.4
3

2
.7
7
B
b
±

0
.0
9

3
.7
2
A
ab

±
0
.3
1

3
.7
5
A
ab

±
0
.2
0

3
.9
3
A
a
±

0
.1
8

%
F
C

6
0
.5
0
A
a
±

4
.2

5
3
.5
0
A
ab

±
4
.2

4
6
.0
0
A
ab

±
5
.3

4
2
.0
0
A
b
±

4
.1

3
7
.0
0
B
a
±

1
.2

3
6
.5
0
B
a
±

2
.6

4
6
.5
0
A
a
±

2
.6

4
1
.5
0
A
a
±

2
.9

S
N
S

2
5
.0
0
A
a
±

0
.7

2
1
.5
0
A
ab

±
1
.3

2
2
.2
5
A
ab

±
1
.2

1
8
.5
0
A
b
±

2
.7

9
.0
0
B
b
±

1
.2

2
0
.7
5
A
a
±

2
.1

1
5
.7
5
B
a
±

1
.1

2
0
.5
0
A
a
±

1
.4

W
N
S

1
1
.2
5
A
a
±

1
.3

9
.0
0
A
a
±

1
.2

1
0
.0
0
A
a
±

1
.7

6
.2
5
A
a
±

0
.6

1
0
.7
5
A
a
±

1
.0

7
.7
5
A
a
±

1
.1

9
.7
5
A
a
±

2
.2

9
.0
0
A
a
±

1
.4

L
S

1
1
.7
1
A
a
±

2
.1

7
.8
5
A
b
±

0
.3
3

9
.6
8
A
ab

±
0
.4
2

8
.8
7
A
ab

±
0
.9
3

1
.2
2
B
b
±

0
.0
5

5
.6
0
A
a
±

0
.4
6

4
.2
6
B
ab

±
0
.3
8

5
.4
7
B
a
±

0
.4
5

L
R

8
.7
2
A
c
±

0
.8

1
4
.5
6
A
ab

±
1
.9

1
0
.6
0
A
b
c
±

0
.4

1
8
.5
8
A
a
±

1
.4

2
.8
6
B
a
±

0
.1
7

6
.8
8
B
a
±

2
.1

6
.1
2
B
a
±

0
.8
2

4
.2
0
B
a
±

0
.5
3

L
T

2
0
.4
4
A
b
±

2
.3

2
2
.4
1
A
ab

±
2
.0

2
0
.2
9
A
b
±

0
.7
8

2
7
.4
5
A
a
±

2
.2
4

4
.0
9
B
b
±

0
.1
8

1
2
.4
9
B
a
±

2
.3

1
0
.4
0
B
ab

±
1
.2

9
.6
8
B
ab

±
0
.9
3

D
M
S

0
.0
1
0
A
b
±

0
.0
0
1

0
.0
3
3
A
ab

±
0
.0
0
2

0
.0
4
2
A
a
±

0
.0
0
1

0
.0
4
0
A
ab

±
0
.0
0
2

0
.0
0
8
A
c
±

0
.0
0
0
7

0
.0
4
0
A
a
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Mitigating effects of acetylcholine supply on soybean seed germination under osmotic stress 621

123



suggests an interesting possibility on the mechanism of

action of Ach and its involvement with auxin (Baluska

et al. 2005). Some experiments have shown that Ach

combined with auxin increases the expression of the

expansin LeEXPA2 gene in the hypocotyls of tomatoes,

when compared to auxin and Ach treatments separately

(Di-Sansebastiano et al. 2014). Expansins are known to be

involved in cell elongation, being codified by a multigene

family, where LeEXPA2 is included, which are regulated

by auxin in the growing regions of young tomato hypocotyl

(Caderas et al. 2000). Di-Sansebastiano et al. (2014) ana-

lyzed the expression of the expansin gene LeEXPA2 in

tomato hypocotyls to investigate the effect of auxin and

Ach in cell elongation. A low level of LeEXPA2 tran-

scription in the treatment only with Ach was observed, but

there was a significant increase in the transcription of

expansins in the Ach treatments when combined with

auxin.

In the results presented herein, there was no increase in

LS under any of the tested concentrations of Ach under

well-water conditions (Fig. 1). This result may be related

to the concentrations used in the study. The literature

provides no support for a clear definition of an optimal

concentration of Ach to stimulate plant growth (Di-

Sansebastiano et al. 2014). According to Lawson et al.

(1978), the concentration of Ach 0–750 lM results in an

inhibitory effect on elongation of apical coleoptile in wheat

(Avena sativa). However, Mukherjee (1980) tested higher

concentrations of Ach in soybean seed germination,

observing a stimulating effect on the elongation of hypo-

cotyls. Parsaeimehr et al. (2015) observed that an exoge-

nous application of 5 lg L-1 of Ach in Chlorella

sorokiniana has a stimulatory role in the growth and

accumulation of lipids. On the other hand, in Vigna ses-

quipedalis, there was suppression in the growth of hypo-

cotyl and, simultaneously, there was an increase in epicotyl

(Hoshino 1983).

Water stress induced by mannitol (-1.0 MPa) signifi-

cantly affected the traits analyzed regarding germination

and seedling development (Table 1). These results are due

to the reduction in water potential of the plant caused by

mannitol, which reduces the water availability to seeds,

promoting the reduction in enzyme activity, generating less

meristematic development and thus reducing speed and the

germination rate (Popinigis 1985; Avila et al. 2007).

The results from the application of Ach in the seeds

germinated under osmotic stress have showed a remarkable

mitigating effect of all tested concentrations of Ach, in

relation to inhibition caused by the water potential of

-1.0 MPa. A concentration 0.5 mM of Ach showed the

best effect on the LS and LR, promoting greater dry matter

accumulation (Table 1). According to Murch (2006),

increased growth and cell wall thickness occur because

Ach is synthesized in the stems, leaves and root nodes in

response to stress. Sugiyama and Tezuka (2011) analyzed

the application of Ach in radish seeds, obtaining as a result

a small effect on the fresh weight of the roots, while

approximately a doubling of dry weight was observed. Ach

exhibits an ability to increase the translocation of carbo-

hydrates, such as sugars and starch, from the cotyledons to

the roots, providing a higher elongation of roots and also

higher biomass accumulation (Sugiyama and Tezuka

2011). Thus, Sugiyama and Tezuka (2011) suggested that

Ach can activate some metabolic systems such as the

glycolytic pathway in the cytoplasm, the TCA cycle and

the electron transport system, as well as metabolic systems

participating in increasing the amount of NAD, DNA and

proteins. The mitigating effect of Ach found in this study is

supported by other studies in the literature. Momonoki and

Momonoki (1993), Roschina (2001) and Horiuchi et al.

(2003) observed similar effects of Ach on various plants

subjected to stress, suggesting it plays an important role in

the response to environmental stimuli (Yamamoto and

Momonoki 2012). These studies support the former

observations of Kostir et al. (1965), suggesting that

exogenous application of Ach influences the germination

and the initial stage of development and growth of plants.

The Ach, the enzyme that hydrolyzes the Ach (AchE—

acetylcholinesterase) and the Ach receptors (nicotinic, such

as nAChRs, and muscarinic, such as mAChR) have been

widely recognized in higher plants (Roschina 2001). In

plants, although the genes encoding the Ach receptors

(AchR) have not been identified, the named ‘‘binding sites

of Ach’’ were found at chloroplasts (Roschina 2001) and at

tonoplast (Gong and Bisson 2002). The Ach-mediated

processes in plants can induce depolarization of the

membranes, affecting the cell-to-cell transport of hormones

and other substances (Momonoki 1997). The AchE gene

was identified in Salicornia europaea L. by RT-PCR using

primers from cloned maize genes, showing that the activity

of AchE increased in the root due to the accumulation of

Na? and Cl-. Therefore, it was proposed that Ach-medi-

ated processes in Salicornia europaea may act in the

elimination of excess salt in the epidermal cells of roots by

improving cell-to-cell transport (Yamamoto et al. 2009).

As observed in our results, Ach promoted growth and

dry matter accumulation in soybean seedlings under

osmotic stress conditions, acting as a stress-mitigating

agent, indicating that Ach must be involved in various

metabolic processes of the plants. Czerpak et al. (2003)

reported that Ach and taurine (a, chemical analogue of

Ach) significantly stimulated the synthesis of some

metabolites in Chlorella vulgaris Beijerinck, particularly

monosaccharides and soluble proteins. Monosaccharides

and soluble proteins may have an important role in osmotic

adjustment in plants subjected to water stress (Morgan

622 I. Braga et al.

123



1984; Manavalan et al. 2009), which could explain, at least

in part, the osmotic stress mitigation effects observed in

our study.

Acknowledgements The authors acknowledge the funding Programa

Institucional de Bolsas de Iniciação Cientı́fica (PIBIC) and Comissão

de Aperfeiçoamento de Pessoal do Nı́vel Superior (CAPES). M.D.

Pissolato is scholarship PIBIC and I. Braga scholarship of doctorate

CAPES.

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	Mitigating effects of acetylcholine supply on soybean seed germination under osmotic stress
	Abstract
	Introduction
	Materials and methods
	Results
	Discussion
	Acknowledgements
	References