derivatives, are polar molecules that do not readily diffuse across
membranes, and thus require an active transport mechanism to
cross them.
Two main classes of plant transmembrane transporters have
been associated with secondary metabolite transport:
ATP-binding cassette (ABC) and multidrug and toxic compound
extrusion (MATE) transporters. However, both these classes have
also been shown to be involved in plant hormone transport. ABC
transporters are localized at the plasma membrane, the tonoplast,
chloroplasts, mitochondria, and peroxisomes and mediate active
transport driven by ATP hydrolysis [5]. MATE transporters, on
the other hand, have been reported to be localized to the tonoplast,
the plasma membrane or the chloroplast and are driven by a transmembrane proton gradient [6]. ABC transporters participate in the
translocation of the apocarotenoid hormones abscisic acid (ABA)
and strigolactone. In Arabidopsis thaliana, two plasma membranelocalized ABC transporters, AtABCG40 and AtABCG30, are
responsible of the import of ABA from the apoplast into the cytosol
[7, 8], and two, ABCG25 and ABCG31 of the export from xylem
parenchyma cells or from the seed coat [8, 9]. A third ABC transporter, AtABCC2 is involved in the vacuolar sequestration of ABA
glucosyl ester (ABA-GE) [10]. In Petunia hybrida the ABC transporter PDR1 is involved in strigolactone exudation from roots
[11]. To date, no transport of apocarotenoids by a MATE transporter has been demonstrated.
Two different experimental approaches are available for the
characterization of transmembrane transporters: reverse genetics,
by knocking out or overexpressing the corresponding gene and
observing the phenotype [11, 12]; this approach can be complemented by isolating organelles such as chloroplasts, mitochondria
or vacuoles from wild-type plants and the corresponding mutants
and/or overexpressors and performing transport experiments, usually with the silicon oil technique [10, 13]. A second approach is
heterologous expression in animal, plant or yeast cells, followed by
observation of transport in intact cells or by isolation of membrane
vesicles [12, 14, 15], that can be used either directly or can be
further fractionated to enrich a specific membrane-type
[13]. Depending on the transport direction, transport activity will
be determined using either outside-out or inside-out vesicles.
While transport by plant ABC and MATE transporters has been
characterized for a series of compounds for which purified substrates are commercially available (for example nicotine, flavonoids,
folates, etc., [5, 16–18]), only few apocarotenoids are commercially
available as standards. To overcome this problem, a complex crude
plant extract can be used to perform transport assays and the uptake
of several metabolites can be evaluated simultaneously by liquid
chromatography-photodiode array–high resolution mass spectrometry (LC-PDA-HRMS) (Fig. 1). This approach has been
90
Olivia Costantina Demurtas et al.
membranes, and thus require an active transport mechanism to
cross them.
Two main classes of plant transmembrane transporters have
been associated with secondary metabolite transport:
ATP-binding cassette (ABC) and multidrug and toxic compound
extrusion (MATE) transporters. However, both these classes have
also been shown to be involved in plant hormone transport. ABC
transporters are localized at the plasma membrane, the tonoplast,
chloroplasts, mitochondria, and peroxisomes and mediate active
transport driven by ATP hydrolysis [5]. MATE transporters, on
the other hand, have been reported to be localized to the tonoplast,
the plasma membrane or the chloroplast and are driven by a transmembrane proton gradient [6]. ABC transporters participate in the
translocation of the apocarotenoid hormones abscisic acid (ABA)
and strigolactone. In Arabidopsis thaliana, two plasma membranelocalized ABC transporters, AtABCG40 and AtABCG30, are
responsible of the import of ABA from the apoplast into the cytosol
[7, 8], and two, ABCG25 and ABCG31 of the export from xylem
parenchyma cells or from the seed coat [8, 9]. A third ABC transporter, AtABCC2 is involved in the vacuolar sequestration of ABA
glucosyl ester (ABA-GE) [10]. In Petunia hybrida the ABC transporter PDR1 is involved in strigolactone exudation from roots
[11]. To date, no transport of apocarotenoids by a MATE transporter has been demonstrated.
Two different experimental approaches are available for the
characterization of transmembrane transporters: reverse genetics,
by knocking out or overexpressing the corresponding gene and
observing the phenotype [11, 12]; this approach can be complemented by isolating organelles such as chloroplasts, mitochondria
or vacuoles from wild-type plants and the corresponding mutants
and/or overexpressors and performing transport experiments, usually with the silicon oil technique [10, 13]. A second approach is
heterologous expression in animal, plant or yeast cells, followed by
observation of transport in intact cells or by isolation of membrane
vesicles [12, 14, 15], that can be used either directly or can be
further fractionated to enrich a specific membrane-type
[13]. Depending on the transport direction, transport activity will
be determined using either outside-out or inside-out vesicles.
While transport by plant ABC and MATE transporters has been
characterized for a series of compounds for which purified substrates are commercially available (for example nicotine, flavonoids,
folates, etc., [5, 16–18]), only few apocarotenoids are commercially
available as standards. To overcome this problem, a complex crude
plant extract can be used to perform transport assays and the uptake
of several metabolites can be evaluated simultaneously by liquid
chromatography-photodiode array–high resolution mass spectrometry (LC-PDA-HRMS) (Fig. 1). This approach has been
90
Olivia Costantina Demurtas et al.