Characterisation of the membrane transport of pilocarpine in cell suspension cultures of Pilocarpus microphyllusby Nathalia Luiza Andreazza, Ilka Nacif Abreu, Alexandra Christine Helena Franklan Sawaya, Paulo Mazzafera

Journal of Plant Physiology


Physiology / Agronomy and Crop Science / Plant Science


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Accepted Manuscript

Title: Characterisation of the membrane transport of pilocarpine in cell suspension cultures of Pilocarpus microphyllus

Author: Nathalia Luiza Andreazza Ilka Nacif Abreu

Alexandra Christine Helena Frankland Sawaya Paulo


PII: S0176-1617(14)00311-3


Reference: JPLPH 52067

To appear in:

Received date: 3-7-2014

Revised date: 27-10-2014

Accepted date: 13-11-2014

Please cite this article as: Andreazza NL, Abreu IN, Sawaya ACHF, Mazzafera

P, Characterisation of the membrane transport of pilocarpine in cell suspension cultures of Pilocarpus microphyllus, Journal of Plant Physiology (2014),

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Ac ce pte d M an us cri pt 1

Characterisation of the membrane transport of pilocarpine in 1 cell suspension cultures of Pilocarpus microphyllus2 3

Nathalia Luiza Andreazza, Ilka Nacif Abreu, Alexandra Christine Helena 4

Frankland Sawaya, Paulo Mazzafera*5 6 †Departamento de Biologia Vegetal, Instituto de Biologia, CP 6109, 7

Universidade Estadual de Campinas, 13083-970, Campinas, SP, Brazil.8 9 *Corresponding author10 e-mail address: 11

Tel: +55 19 35216213 Fax: ++55 19 35216185 12 13

Running head title: Membrane transport of pilocarpine in Pilocarpus 14 microphyllus cells.15 16


Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with 18 important pharmaceutical applications. Previous reports have investigated the 19 production of pilocarpine by P. microphyllus cell cultures and tried to establish 20 the alkaloid biosynthetic route. However, the site of pilocarpine accumulation 21 inside of the cell and its exchange to the medium culture is still unknown. 22

Therefore, the aim of this study was to determine the intracellular 23 accumulation of pilocarpine and characterise its transport across membranes 24 in cell suspension cultures of P. microphyllus. Histochemical analysis and 25

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Ac ce pte d M an us cri pt 2 toxicity assays indicated that pilocarpine is most likely stored in the vacuoles 1 probably to avoid cell toxicity. Assays with exogenous pilocarpine 2 supplementation to the culture medium showed that the alkaloid is promptly 3 uptaken but it is rapidly metabolised. Treatment with specific ABC protein 4 transporter inhibitors and substances that disturb the activity of secondary 5 active transporters suppressed pilocarpine uptake and release suggesting that 6 both proteins may participate in the traffic of pilocarpine to inside and outside 7 of the cells. As Bafilomicin A1, a specific V-type ATPase inhibitor, had little 8 effect and NH4Cl (induces membrane proton gradient dissipation) had 9 moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) 10 strongly inhibited the transport of pilocarpine, it is believed that ABC proteins 11 play a major role in the alkaloid transport across membranes but it is not the 12 exclusive one. Kinetic studies supported these results.13 14

Key words: Pilocapus microphyllus, pilocarpine, ABC proteins, subcellular 15 localization, cell suspension, transport. 16 17 18 19

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Ac ce pte d M an us cri pt 3

Introduction1 2

The growth of cell suspension cultures using bioreactors is a promising 3 technique to produce secondary metabolites of economical importance4 (Kolewe et al., 2008; Ruffoni et al., 2010; Hussain et al., 2012). It has several 5 advantages, such as the ability to control the medium conditions by the 6 addition of compounds that stimulate production of the molecule of interest. 7

This technology is particularly important once the success of the culture 8 depends on the demand for the target molecule, its price in the market, the 9 culture yield and the culture costs. The culture costs are intimately related to 10 culture yield, thus relatively low costs might be obtained if the compound is 11 released in the medium and easily recovered by medium replacement12 (Verpoorte et al., 1993, 2009; Sakai et al., 2002). Therefore, it is essential to 13 understand the processes involved in the transport of metabolites across 14 membranes and their accumulation inside the cell.15

Different protein families have been characterised regarding secondary 16 metabolites transport across plant cell membranes: ATP-binding cassette 17 proteins (ABC transporters) are implicated in the transport of a multitude of 18 substrates and also associated with detoxification processes (Kang et al., 19 2011); MATE (multidrug and toxic compound extrusion) are typically involved 20 in detoxification process (Omote et al., 2006; Yasaki et al., 2008); NRT (nitrate 21 transport) transports dipeptides, tripeptides and nitrate (Tsay et al., 2007; 22

Nour-Eldin and Halkier, 2013); and PUP (purine uptake permease) is primary 23 metabolite transporter with an additional role in secondary metabolites 24 translocation (Jelesko, 2012; Carqueijeiro et al., 2013). H+-ATPase (V-25

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Ac ce pte d M an us cri pt 4

ATPase, P-ATPase, F1/F0-ATPase) and H +-pyrophosphatase (V-PPase, H+-1

PPase) are indirectly involved in the secondary metabolites transport, since2 they are responsible for creating a proton gradient across the membrane, 3 which is necessary for the transport mediated by other intrinsic protein 4 (Gaxiola et al., 2007; Rea, 2007).5

In Catharanthus roseus, at least five cell compartments are involved in 6 the production of monoterpene indole alkaloids, and intermediates of the 7 biosynthesis pathway are transported by these compartments, through 8 different transport mechanisms, where they serve as substrates for specific 9 enzymes (Facchini and De Luca, 2008; Yazaki et al., 2008). 10