Acetylcholine causes rooting in leaf explants of in vitro raised tomato

(Lycopersicon esculentum Miller) seedlings

Kiran Bamel, Shrish Chandra Gupta, Rajendra Gupta

Department of Botany, University of Delhi, Delhi-110007, India

Received 1 November 2006; accepted 24 January 2007
Abstract
The animal neurotransmitter acetylcholine (ACh) induces rooting and promotes secondary root formation in leaf explants of tomato
(Lycopersicon esculentum Miller var. Pusa Ruby), cultured in vitro on Murashige and Skoog's medium. The roots originate from the midrib of leaf
explants and resemble taproot. ACh at 10
5
M was found to be the optimum over a wide range of effective concentrations between 10
7
and
10
3
M. The breakdown products, choline and acetate were ineffective even at 10
3
M concentration. ACh appears to have a natural role in
tomato rhizogenesis because exogenous application of neostigmine, an inhibitor of ACh hydrolysis, could mimic the effect of ACh. Neostigmine,
if applied in combination with ACh, potentiated the ACh effect.
2007 Elsevier Inc. All rights reserved.
Keywords: Acetylcholine; Acetylcholinesterase; Lycopersicon esculentum; Neostigmine; Non-neuronal; Rhizogenesis; Tomato
Introduction
Acetylcholine (ACh) and its metabolizing enzymes are
ubiquitously present in animals as well as in plants (Horiuchi
et al. 2003). The role of ACh as a neurotransmitter in animals is
well-established (Hoffman and Taylor, 2001). However, in
recent years increasing evidence has accumulated to suggest
non-neuronal functions for ACh, e.g. in regulation of
morphogenetic cell movements, cell proliferation, growth and
differentiation (Lauder and Schambra, 1999; Soreq and Seid-
man, 2001; Wessler et al., 2001; Cousin et al., 2005). In plants,
ACh affects changes in electric potentials as well as several
physiological functions (see for reviews Tretyn and Kendrick,
1991; Roshchina, 2001). However, no investigation has yet
been reported concerning the effect of acetylcholine on
morphogenesis in explants cultured in vitro. In the present
study, we investigated the effect of acetylcholine and its
breakdown products, choline and acetate, as well as neostig-
mine (Nst), an inhibitor of ACh breakdown, on morphogenic
behaviour of leaf explants of tomato cultured on Murashige and
Skoog's (MS) medium (Murashige and Skoog, 1962). Tomato
was chosen as an experimental system because (a) its
morphogenic behaviour in vitro is well known (Sink and
Reynolds, 1986), and (b) the tomato family, Solanaceae
contains ACh, the ACh hydrolysing enzyme acetylcholinester-
ase (AChE) as well as several alkaloids that are known to affect
ACh system in animals (Roshchina, 2001).
Materials and methods
Plant material
Seeds of tomato, Lycopersicon esculentum Miller var. Pusa
Ruby (National Seeds Corporation, New Delhi) were soaked in
distilled water for an hour, surface sterilized with 1% (v/v)
Polysan (Polypharma, Mumbai), and 0.1% (w/v) HgCl
2
(E.
Merck, Mumbai), followed by rinsing with rectified spirit and
sterilized distilled water. Seeds were germinated on Knop's
medium containing 3% sucrose and 0.8% agar. The pH of the
medium was adjusted to 5.8 before autoclaving at 121 C for
15 min. at 1.02 kg/cm
2
. For the in vitro culture of plants, leaves
from 30-day-old in vitro raised seedlings were used in all the
experiments. Only the top two (distal) leaves were cultured.
Life Sciences 80 (2007) 23932396
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doi:10.1016/j.lfs.2007.01.039
Leaves (approximately 0.5 cm) were trimmed at margins and
cultured on MS basal medium alone as well as supplemented
individually with 10
9
to 10
3
M ACh, choline, acetate or Nst.
ACh and Nst were filter-sterilized. The media contained 3%
sucrose and 0.8% agar. All the cultures were maintained at 28
2 C under white fluorescent light of 450460 W cm
2
programmed for 16 h photoperiod for 4 weeks. Explants in
culture were subjected to chemical treatment from the time of
inoculation. For each treatment, 48 explants were cultured and
experiments were repeated thrice. The data of observation after
30 days of culture/continuous chemical treatment are reported
here. Each datum represents an average of three experiments
with 144 explants per treatment. The data were analyzed
statistically and expressed as the meanSE (within 95%
confidence limit).
Estimation of activity of acetylcholinesterase (AChE)
Tomato cultures (4 g) were crushed in liquid nitrogen and
homogenized in 8 ml of 4% (w/v) ammonium sulphate in 0.1 M
K-Pi buffer (pH 7; 1:2 w/v); the homogenate was stirred for
20 min, filtered through cheesecloth; centrifuged at 12,000 g for
15 min at 4 C. The supernatant was concentrated to dried
powder form by lyophilisation and the proteins re-suspended in
a small volume (2 ml) of 0.1 M K-Pi buffer, pH 7 and desalted
by employing gel filtration on Sephadex G-25. Protein rich
fractions were pooled, and tested for AChE activity (Ellman
et al., 1961).
Statistical analysis of results
Data was subjected to univariate analysis of variance.
Significant differences were established using post-hoc Tukey's
HSD test.
Results
ACh (10
7
to 10
3
M) and neostigmine (10
7
to 10
3
M)
induced rooting in excised leaves of L. esculentum var. Pusa
Ruby cultured in vitro and promoted the number of roots per
explant as well as formation of secondary roots (Figs. 1 and 2).
A one-way ANOVA of data revealed that the there was
significant induction of rooting as well as formation of
secondary roots in explants treated with ACh or neostigmine
(Fig 1A, B; Fig 2A, B). ACh was ineffective at 10
8
and
10
9
M. The optimum levels of ACh were 10
5
M for root
induction (Fig. 1A), 10
4
M for increasing the number of roots
(Fig. 1C), and 10
6
M for induction of secondary roots
(Fig. 1B, D). Roots were always formed at the base of the
midrib. Our tests showed that tomato leaves have AChE activity
(578 pmol ATChI hydrolysed/mg protein/s) which is compa-
rable to that present in nerves of lower animals. Naturally, the
Fig. 1. AD. Effect of ACh on morphogenic responses of leaf explants of
tomato, Lycopersicon esculentum var. Pusa Ruby. ACh (10
9
to 10
3
M) was
provided continuously in the culture medium from the first day of culture for
30 days. Leaf explants used here were excised from 30-day-old seedlings raised
in vitro on MS basal medium. The data presented here are an average of three
experiments with 144 explants per treatment. Error bars showS.E.M.

Denotes significant differences between treatment and control (P0.05);

highly significant (P0.01);

very highly significant (P0.001). A.
Percentage of leaf explants forming roots. B. Percentage of roots forming
secondary roots. C. Average number of roots formed per responding explant. D.
Average number of secondary roots developed per root.
2394 K. Bamel et al. / Life Sciences 80 (2007) 23932396
AChE activity would result in formation of choline and acetate
in culture medium. However, very high concentrations
(10
3
M) of choline chloride or potassium acetate did not elicit
any rooting response. Treatment of cultures with Nst, an
inhibitor of ACh breakdown, caused rooting in leaf explants
with optimum response at 10
4
M Nst for primary root and
10
3
M for secondary roots (Fig. 2). Neostigmine (10
4
M)
caused 76% inhibition of AChE in cultured leaves (decrease to
138 from a high of 578 pmol ATChI hydrolysed/mg protein/s in
controls). When Nst was supplemented to MS medium in
different combinations with ACh, Nst potentiated the effect of
ACh (64% explants formed roots on 10
4
M ACh+10
3
M Nst
as compared to 14% on ACh alone. Interestingly, it was also
observed that ACh and Nst inhibited callusing on leaf explants
that is otherwise considered a frequent feature in plant tissue
cultures.
Discussion
Rooting in plants (in vivo as well as in vitro) is a classic
response to the plant hormone auxin. The effectiveness of ACh
or inhibitors of its breakdown in causing rooting raises an
interesting possibility about the mechanism of action of ACh
and its involvement in auxin action. At present, we have no
estimate for the endogenous concentration of ACh in tomato
leaves or of the concentration required endogenously to cause
rhizogenesis. However, we have reason to believe that the ACh
is a natural regulator of rooting in tomato and that the
concentration of ACh required endogenously would be much
lower than reported here to be effective for exogenous
application (10
7
10
3
M, optimum being 10
5
M) because:
(a) ACh (10
7
10
3
M) has rhizogenic effect, (b) AChE is
present in tomato leaves and hydrolyses ACh, (c) choline, the
breakdown product/precursor of ACh has no effect, (d) AChE
inhibitor Nst inhibits AChE in tomato cultures and it also
simulates the effect of ACh, and (e) Nst potentiates the ACh
effect. It would be interesting to find whether ACh at such lower
concentrations would be acting like a growth hormone or
interacting with auxin or other hormones. Asymmetric
distribution of AChE in coleoptiles of gravistimulated seedlings
of maize and inhibition of gravity response by inhibiting AChE
by Nst has been demonstrated (Momonoki, 1997). Alternative-
ly, one must check the possibility that the effects reported here
could be due to non-catalytic effects of AChE rather than of
ACh. It has been shown in some studies on animal
differentiation and developments that chronic exposure of
developing cells to ACh/AChE induces amplification of AChE
gene and overproduction of the enzyme, and that AChE also
performs non-catalytic role as a mediator of cell adhesion, cell
movement, and differentiation (Soreq and Zakut, 1993;
Charpentier et al., 1998; Grisaru et al., 1999; Sharma et al.,
2001; Farchi et al., 2003). The tomato leaf cultures used in the
Fig. 2. AD. Effect of neostigmine on morphogenic responses of leaf explants of
tomato, Lycopersicon esculentum var. Pusa Ruby. Neostigmine (10
9
to
10
3
M) was provided continuously in the culture medium from the first day of
culture for 30 days. Leaf explants used here were excised from 30-day-old
seedlings raised in vitro on MS basal medium. The data presented here are
an average of three experiments with 144 explants per treatment. Error bars
showS.E.M.

Denotes significant differences between treatment and control
(P0.05);

highly significant (P0.01);

very highly significant
(P0.001). A. Percentage of leaf explants forming roots. B. Average number
of roots induced per responding explant. C. Percentage of roots developing
secondary roots. D. Average number of secondary roots formed per root.
2395 K. Bamel et al. / Life Sciences 80 (2007) 23932396
present study may also be overproducing AChE after exposure
to ACh/AChE inhibitors for 30 days because AChE in leaf
explants is inhibited only to about 76% by 10
4
M Nst although
100 times lesser concentration (10
6
M) is enough to cause
complete inhibition of partially purified AChE from tomato
leaves (unpublished observations in M. Phil. Thesis of P.
Kavita, Delhi University 2002).
Conclusion
The observations presented here provide the first in vitro
experimental evidence for the natural role of ACh in
differentiation and morphogenesis in a plant. Further studies
would be required to find out (a) the mechanism by which ACh
interacts with the plant hormone auxin's classical role in
rooting, and (b) common features in morphogenetic roles of
ACh in plants and animals.
Acknowledgements
SRF to KB from CSIR, Indo-Israel grant from DST to SCG
and grant from UGC to RG.
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