Analytical and Fluorimetric Methods for the Characterization of the Transmembrane

Transport of Specialized Metabolites in Plants

Abstract

The characterization of membrane transport of specialized metabolites is essential to

understand their metabolic fl uxes and to implement metabolic engineering strategies towards
the production of increased levels of these valuable metabolites. Here, we describe a set of
procedures to isolate tonoplast membranes, to check their purity and functionality, and to
characterize their transport properties. Transport is assayed directly by HPLC analysis and
quantifi cation of the metabolites actively accumulated in the vesicles, and indirectly using the
pH sensitive fl uorescent probe ACMA (9-amino-6- chloro-2-methoxyacridine), when a proton
antiport is involved.

Key words ABC transporters , ACMA , Alkaloids , BY-2 cells , Catharanthus roseus, H +-antiport ,
MATE transporters , Tobacco , Tonoplast vesicles , Transmembrane transport , Transmembrane
pH gradient , Uptake assay

Introduction

Plants exhibit a unique metabolic plasticity implicated in many aspects of their environmental
success as sessile organisms. For humans, this specialized metabolism provides a wealth of
natural products with innumerous applications as medicines, colorants, fl avors, fragrances,
cosmetics, etc. Currently, next generation sequencing is fuelling a burst in molecular knowledge
on specialized pathways, driving intense research seeking to obtain increased yields of
metabolites for commercial exploitation or improvement of plant defenses. However, in spite
of intensive investigation, little is known about the transmembrane transportmechanisms
involved in specialized pathways, even if they often involve organ/tissue/ cell translocation
events as well as subcellular compartmentation (e.g., nicotine, berberine, morphine, and
vinblastine pathways) [ 1].

Therefore, membrane transport of specialized metabolites is emerging as a newly developing

research line, essential to the understanding of metabolic fl uxes and to the implementation of
metabolic engineeringstrategies aimed at increased levels of valuable metabolites. Two major
mechanisms have been implicated in the transmembrane transportof specialized metabolites in
plants: primary transport mediated by ABC (ATP-binding cassette) transporters and secondary
transport mediated by MATE(multidrug and toxic compound extrusion) transporters functioning
as H +-antiport s [ 1, 2]. Both have been implicated in the transport of alkaloids and fl avonoids,
and ABC transportershave been further implicated in the transport of terpenoids, ABA,
strigolactones and lipid and monolignol deposition in the extracellular surface [ 1, 2]. The
isolation of pure and functional organelles or organellederived membrane vesicles is a
prerequisite for successful transport studies, with the membrane sidedness being a key issue in
the case of isolated vesicles. The determination of the radioactivity entrapped by the
cells/organelles/membrane vesicles upon incubation with a radiolabeled substrate is a reliable
and sensitive method to measure uptake [ 3], but its main drawback lies in the fact that the
majority of alkaloids or other specialized metabolites are not commercially available as
radioisotopes. On the other hand, in some cases, the transported solute may be quantifi ed by
highly sensitive analytical methods, including HPLC[ 4]. Uptake studies relying on
electrophysiological measurements and specifi c fl uorescent probes, sensitive for instance to
the membrane potential or pH, are also widely reported [ 5]. Thus, the pH sensitive fl uorescent
probe ACMA(9-amino-6-chloro-2-methoxyacridine) has been frequently used to measure
changes in vacuolar pH when a specifi c substrate crosses the tonoplast through a putative H
+/solute antiport system ( see Note 1) [ 5]. Important issues when using membrane vesicles in
transport experiments are the tightness and orientation of the membrane. While sealing may
be optimized by fusing the biological membranes with liposomes, the manipulation of the
orientation (right side out or inside-out) may be useful depending on both the type and
localization of the transport system under study. Thus, the production of inverted vesicles from
isolated plasma membranes (cytoplasmic side-out) may be achieved with Brij 58 [ 6]. This vesicle
system is appropriate to study H +- dependent antiport systems after exogenous addition of ATP
that promotes the acidifi cation of the vesicle lumen through the activation of P-ATPase. A fi rst
good evidence for the involvement of a mediated transport system comes from the observation
that the initial velocities of transport, over a specifi c range of substrate concentrations, follow
a saturation kinetics, providing at the same time useful information about the kinetic
parameters [ 7]. The simultaneous addition of the transported substrate and a putative
competitor or inhibitor may allow the characterization of the specifi city of the transport system
[ 3, 8]. The thermodynamic aspects (energetics) involved in the substrate uptake may be
experimentally evaluated by studying the H + (or Na +) dependency of the transport system, by
measuring, for instance, the uptake at different pH values, the effect of protonophores, or the
involvement of a primary transport system, usually through the addition of ATP or PPi [ 5, 8].
Here, we describe the methodology used to characterize the vacuolar transport of the medicinal
alkaloidsfrom Catharanthus roseus [ 4], which was also used to study the transport of nicotine
in tonoplast membranes from tobaccoleaves and tobacco BY-2 cells . This approach, which may
be easily adapted to study the transport of other specialized metabolites in other plant models,
involves the following steps:

1. Isolation of tonoplast vesicles . 2. Characterization of the purity of tonoplast vesiclesby

Western blot and assay of vacuolar (V)-H +-ATPase hydrolytic activity by measurement of ATP
hydrolysis in the presence of azide, a mitochondrial ATPase inhibitor, and vanadate, a plasma
membrane ATPase inhibitor ( see Note 2). 3. Evaluation of membrane functionality and integrity
by demonstrating the generation of a transtonoplast pH gradient by an active V-H +-ATPase
and/or an active V-H +-pyrophosphatase (PPase) ( see Note 3). 4. Assay of the uptake of alkaloids
by tonoplast vesiclesfollowed by recovery of tonoplast vesiclesby ultracentrifugation,
alkaloidextraction, and analysis by HPLC -DAD. This assay is performed in the presence of an ABC
transporter inhibitor and in the presence of agents disturbing the transmembrane pH gradientto
establish the nature of the transporter involved. 5. Spectrofl uorometric, indirect assay of
transport using the pHsensitive probe ACMA , since a dependence on the transmembrane pH
gradientwas observed.

Materials

Plant Materials

1. Plants of C. roseus (L.) G. Don cv. Little Bright Eye are grown at 25 °C, under a 16 h photoperiod,
using light with a maximum intensity of 70 μmol m -2 s -1. Seeds are acquired from AustraHort
(Australia) and voucher specimens are deposited at the Herbarium of the Department of Biology
of the Faculty of Sciences of the University of Porto (PO 61912). Plants used for isolation of
tonoplast vesiclesare 4–8-month-old, fl owering plants. 2. Plants of Nicotiana tabacum cv. Petite
Havana SR1 are grown at 21 °C, under a 12 h light photoperiod, at 35 μmol m -2 s -1. Plants
suitable for isolation of tonoplast vesiclesare 6–8-weekold, nonfl owering plants. 3. Cell
suspension cultures of Nicotiana tabacum (L.) cv. Bright Yellow 2 (BY-2; Riken BRC) are grown at
26.5 °C, in the dark, on an orbital shaker, at 125 rpm, in Linsmaier and Skoog medium composed
of MS macronutrients and micronutrients, 200 mg L -1 KH 2PO 4, 100 mg L -1 myo-inositol, 1 mg
L -1 thiamine·HCl, 0.2 mg L -1 2,4-dichlorophenoxyacetic acid, and 3 % sucrose, pH 5.8. Cells are
subcultured every 7 days by adding 2.5 mL of the full grown culture to 47.5 mL of fresh medium
in 250 mL flasks.

Isolation of Tonoplast Vesicles

The buffers and solutions should be prepared no more than a few days prior to isolation of the
vesicles.

1. Extraction buffer: 250 mM sucrose, 100 mM KCl, 2 mM EDTA, 3 mM MgCl 2, 70 mM Tris–HCl,

pH 8. Filter-sterilize and store at 4 °C. Immediately before use add 1 mM phenylmethylsulfonyl
fl uoride (PMSF; from a 100× stock solution prepared in 100 % ethanol, stored at −20 °C), 0.1 %
(w/v) bovine serum albumin (BSA), and 2 mM dl -dithiothreitol (DTT). BSA and DTT may be
directly added to the buffer or prepared separately in stock solutions, aliquoted and stored at
−20 °C. Aliquots should not be thawed more than once. 2. Resuspension buffer: 15 % (v/v)
glycerol, 1 mM EDTA, 20 mM Tris–HCl, pH 7.5. Filter-sterilize and store at 4 °C. Immediately
before use add 1 mM PMSF, and 1 mM DTT. 3. Solutions for sucrose step gradient (made fresh):
32 % or 46 % (w/v) sucrose, 1 mM EDTA, 20 mM Tris–HCl, pH 7.5. Immediately before use add 1
mM PMSF, and 1 mM DTT. 4. Reagents for protein quantifi cation with Lowry method [ 9]. 5.
Liquid nitrogen. 6. Ultra-Turrax T25 apparatus (IKA, Janke & Kunkel, Germany). 7. Glass potter
homogenizer (40 mL vessel, tight, WHEATON, USA). 8. Cheesecloth. 9. High capacity centrifuge
(10,000 × g). 10. Ultracentrifuge (80,000–100,000 × g) with swing-out rotor. 11. Lyophilizer.

Western Blot

1. 50 mM Tris-HCl, pH 7.5. 2. Reagents for protein quantifi cation with Bradford method [ 10],
10 % acrylamide gels, nitrocellulose membranes. 3. Primary antibodies: ER marker—rabbit anti-
serum raised against calreticulin (a gift from J. Denecke, University of Leeds), at a 1:10,000
dilution, or rabbit anti-serum raised against STM1 (#AS 07266, AGRISERA, Germany) at a 1:1000
dilution; chloroplast marker—rabbit anti-serum raised against the chloroplast inner envelope
TIC 40 protein (#AS 10709-10, AGRISERA, Germany), at a 1:2500 dilution; tonoplast markers—
rabbit anti-sera raised against a V-H +-ATPase (#AS 07213, AGRISERA, Germany) and against a
V-H +-PPase [ 11] (may be requested to Prof. M. Maeshima—maeshima@ agr.nagoya-u.ac.jp),
both at a 1:2000 dilution. 4. Secondary antibody: peroxidase conjugated goat anti-rabbit (Santa
Cruz Biotechnology, Inc) at 1:7500 dilution. 5. Chemiluminescent substrate ECL™ (GE Healthcare,
Lifesciences), water bath.

V-H + -ATPase Hydrolytic Activity

1. Reagents: ATP, sodium azide, sodium orthovanadate (prepared as in Subheading 2.5),

Triton X-100, KCl, sodium molybdate, MgSO 4 in 30 mM Tris-MES pH 8, TCA, perchloric
acid, NaH 2PO 4. 2. Reaction solution: 3 mM ATP, 0.02 % Triton X-100, 50 mM KCl, 1 mM
sodium molybdate, and 6 mM MgSO 4 in 30 mM Tris-MES, pH 8. 3. Stop solution: 10 %
TCA with 4 % perchloric acid. 4. Ames solution [ 12]: 1 volume 10 % ascorbic acid, 6
volumes of 4.2 g ammonium molybdate, and 28.6 mL H 2SO 4 in 1 L H 2O. 5. 10 mM NaH
2PO 4 with 0.02 % Triton X-100. 6. Incubator with agitation. 7. Table top centrifuge (2400
× g). 8. Spectrophotometer (O.D. 820 nm). 9. Glass tubes washed with phosphate-free
solutions (Milli-Q water and ethanol 100 %).
Activity of Proton Pumps

1. Reaction buffer for vesicles isolated from C. roseus and N. tabacum leaves: 30 mM KCl, 50 mM
NaCl, 20 mM Hepes, pH 7.2. Filter-sterilize and store at 4 °C. 2. Reaction buffer for vesicles
isolated from BY-2 cell cultures: 100 mM KCl, 10 mM MOPS-Tris, pH 7.2. Filter-sterilize and store
at 4 °C. 3. 200 mM adenosine 5′-triphosphate dipotassium salt (ATP, A8937 Sigma-Aldrich)
prepared in 200 mM Bis-Tris Propane— this solution will have directly a fi nal pH of 7.2. Prepare
aliquots and store at −20 °C. The aliquots should not be thawed more than once. 4. 20 mM
potassium pyrophosphate (PPi, 32243-1 SigmaAldrich) stock solution prepared in H 2O. Store at
−20 °C. 5. 2 mM ACMA(A5806 Sigma-Aldrich) stock solution prepared in 100 % ethanol. Store at
−20 °C. 6. 500 mM MgCl 2·6H 2O stock solution (Merck) prepared in H 2O. 7. Stock solutions of
inhibitors and uncoupling agents: 150 mM NH 4Cl (Merck), 50 mM KNO 3 (221295, Sigma-
Aldrich), 50 mM sodium azide (S8032 Sigma-Aldrich), 10 mM concanamycin A (27689 Sigma-
Aldrich), 50 mM sodium orthovanadate (S6508 Sigma-Aldrich). 8. Spectrofl uorometer with
excitation and emission wavelengths set at 415 and 485 nm respectively. 9. 2 mL UV spectrofl
uorometer cuvette with small magnetic stirrer.

Uptake Assay

1. Reaction buffers as in Subheading 2.5. 2. Stock solutions: 1 M DTT, 500 mM sodium creatine
phosphate dibasic tetrahydrate (27920 Sigma-Aldrich), 50 mM CCCP (carbonyl cyanide m-
chlorophenyl hydrazine; SigmaAldrich) in methanol; ATP, MgCl 2, inhibitors, and uncoupling
agents stock solutions (Subheading 2.5); specialized metabolites (Subheading 2.7). 3. Creatine
Kinase MM Fraction from human heart (C9983- 100UG Sigma-Aldrich). 4. Ultracentrifuge
(100,000 × g). 5. Gaseous N 2 for sample drying.

Proton Antiport Assay

1. Reaction buffers, ATP, PPi, ACMA , and MgCl 2 stock solutions as in Subheading 2.5. 2.
Specialized metabolites stock solutions prepared in methanol (sonicate if necessary): 100 mM
vindoline (Pierre Fabre), 9 mM catharanthine (Pierre Fabre), 33 mM papaverine (P-3510 Sigma-
Aldrich), 25 mM ajmaline (A-7252 Sigma-Aldrich), 100 mM atropine (A-0132 Sigma-Aldrich), 6.5
mM nicotine (N3876 Sigma-Aldrich). 3. Spectrofl uorometer with excitation and emission
wavelengths set at 415 and 485 nm, respectively. 4. 2 mL UV spectrofl uorometer cuvette with
small magnetic stirrer.

Methods

solation of Tonoplast Vesicles

Tonoplast vesiclesare isolated according to [ 13] and [ 14] with minor modifications. All the
procedures are carried out at 4 °C and all materials are precooled.

1. For plants ( see Note 4), homogenize ~5 g of young fully developed leaves without the midrib
in 100 mL of extraction buffer, using the Ultra-Turrax T25 apparatus, at 13,000 rpm, for pulses
of 20 s, over a period of about 5 min, at 4 °C (precool the beaker at 4 °C for 30 min). During the
homogenization keep the beaker surrounded by ice. Repeat two times to obtain an extract from
15 g of leaves. For BY-2 cells , centrifuge 5 × 50 mL of 5 days old cultures at 1500 × g, for 3 min,
at 4 °C, wash the cells twice with cold Milli-Q water, resuspend in 300 mL of extraction buffer
and homogenize with the Ultra-Turrax as above. 2. Filter the homogenate through four layers of
cheesecloth, centrifuge at 10,000 × g for 10 min, at 4 °C, and collect the supernatant. 3.
Centrifuge the supernatant at 100,000 × g for 1 h, at 4 °C, and release the pellets into a total
volume of 8 mL of resuspension buffer. 4. Transfer the pellets in resuspension buffer to a glass
potter homogenizer and perform ~60 up-and down cycles avoiding the formation of air bubbles,
until the pellets are completely resuspended. 5. Overlay the homogenate on a 32 %/46 %
sucrose step gradient with the relative proportions of 2.5:5:3, and centrifuge at 80,000 × g for 3
h, at 4 °C, in a swing-out rotor, using an average deceleration. 6. Remove the upper layer (0 %)
and then collect the 0 %/32 % interface containing the tonoplast vesiclesand transfer to another
ultracentrifuge tube. 7. Dilute in 4 volumes of resuspension buffer and centrifuge at 100,000 ×
g for 45 min, at 4 °C. 8. Resuspend the membrane pellet in 0.3–1 mL of resuspension buffer
(without PMSF or DTT), aliquot, freeze in liquid nitrogen, and store at −80 °C until use. After
thawing, do not freeze again, as activity will be lost.

Characterization of Vesicles Purity by Western Blot

1. Lyophilize the vesicle samples, reconstitute in 50 mM TrisHCl pH 7.5, and determine protein
concentration using the Bradford method [ 10]. 2. Mix protein samples with SDS-PAGEloading
buffer, heat at 70 °C for 10 min, perform SDS-PAGE in 10 % acrylamide gels and transfer to a
nitrocellulose membrane. Load 10 mg of protein for calreticulin and TIC 40 immuno-detection,
and 1 mg of protein for V-H + -ATPase and V-H + -PPase immuno-detection. 3. Perform the
Western blot and detection using standard protocols ( see Note 5).