Acetylcholine causes rooting in leaf explants of in vitro raised tomato (Lycopersicon esculentum Miller) seedlings

Life Sciences 80 (2007) 2393 – 2396 www.elsevier.com/locate/lifescie

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 Seidman, 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 neostigmine (Nst), an inhibitor of ACh breakdown, on morphogenic behaviour of leaf explants of tomato cultured on Murashige and ⁎ Corresponding author. Tel.: +91 9212204551, +91 11 65460096. E-mail address: [email protected] (R. Gupta). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.01.039

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 acetylcholinesterase (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) HgCl2 (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/cm2. 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.

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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 450–460 μW 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 mean ± SE (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 comparable to that present in nerves of lower animals. Naturally, the Fig. 1. A–D. 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 show ± S.E.M. ⁎Denotes significant differences between treatment and control (P≤ 0.05); ⁎⁎highly significant (P ≤ 0.01); ⁎⁎⁎very highly significant (P ≤ 0.001). 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.

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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

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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). Alternatively, 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. A–D. 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 show ± S.E.M. ⁎Denotes significant differences between treatment and control (P ≤ 0.05); ⁎⁎highly significant (P ≤ 0.01); ⁎⁎⁎very highly significant (P ≤ 0.001). 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.

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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. References Charpentier, A., Villatte, F., Fournier, D., 1998. Acetylcholinesterase increase in Drosophila as a mechanism of resistance to insecticide. In: Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. (Eds.), Structure and Function of Cholinesterases and Related Proteins. Plenum Press, New York, pp. 503–507. Cousin, X., Strähle, U., Chatonnet, A., 2005. Are there non-catalytic functions of acetylcholinesterases? Lessons from mutant animal models. BioEssays 27 (2), 189–200. Ellman, G.L., Courtney, K.D., Andres, V.J., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7 (7), 88–95.

Farchi, N., Soreq, H., Hochner, B., 2003. Chronic acetylcholinesterase overexpression induces multilevelled aberrations in mouse neuromuscular physiology. Journal of Physiology 546 (1), 165–173. Grisaru, D., Sternfeld, M., Eldor, A., Glick, D., Soreq, H., 1999. Structural roles of acetylcholinesterase variants in biology and pathology. European Journal of Biochemistry 264 (3), 672–686. Hoffman, B.B., Taylor, P., 2001. Neurotransmission: the autonomic and somatic motor nervous systems. In: Hardman, J.G., Limbird, L.E. (Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics, tenth ed. McGraw Hill, New York, pp. 115–153. Horiuchi, Y., Kimura, R., Kato, N., Fujii, T., Seki, M., Endo, T., Kato, T., Kawashima, K., 2003. Evolutional study on acetylcholine expression. Life Sciences 72 (15), 1745–1756. Lauder, J.M., Schambra, U.B., 1999. Morphogenetic roles of acetylcholine. Environmental Health Perspective 107 (Supplement), 65–69. Momonoki, Y.S., 1997. Asymmetric distribution of acetylcholinesterase in gravistimulated maize seedlings. Plant Physiology 114 (1), 47–53. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15 (3), 473–497. Roshchina, V.V., 2001. Neurotransmitters in Plant Life. Science Publishers Inc., Enfield, New Hampshire. (Chapter 3). Sharma, K.V., Koenigsberger, C., Brimijoin, S., Bigbee, J.W., 2001. Direct evidence for an adhesive function in the noncholinergic role of acetylcholinesterase in neurite outgrowth. Journal of Neuroscience Research 63 (2), 165–175. Sink, K.C., Reynolds, J.F., 1986. Tomato (Lycopersicon esculentum Mill.). In: Bajaj, Y.P.S. (Ed.), Biotechnology in Agriculture and Forestry, vol. 2. Springer-Verlag, Berlin, pp. 319–344. Soreq, H., Seidman, S., 2001. Acetylcholinesterase — new roles for an old actor. Nature Reviews Neurosciences 2, 294–302. Soreq, H., Zakut, H., 1993. Human Cholinesterases and Anticholinesterases. Academic Press, New York. (Chapter 6). Tretyn, A., Kendrick, R.E., 1991. Acetylcholine in plants: presence, metabolism and mechanism of action. Botanical Review 57 (1), 33–73. Wessler, I., Kilbinger, H., Bittinger, F., Kirkpatrick, C.J., 2001. The biological role of non-neuronal acetylcholine in plants and humans. Japanese Journal of Pharmacology 85 (1), 2–10.