Alternate Energy-Dependent Pathways for the Vacuolar Uptake of Glucose and Glutathione Conjugates

Abstract

Through the development and application of a liquid chromatography-mass spectrometry-based procedure for measuring the transport of complex organic molecules by vacuolar membrane vesicles in vitro, it is shown that the mechanism of uptake of sulfonylurea herbicides is determined by the ligand, glucose, or glutathione, to which the herbicide is conjugated. ATP-dependent accumulation of glucosylated chlorsulfuron by vacuolar membrane vesicles purified from red beet (Beta vulgaris) storage root approximates Michaelis-Menten kinetics and is strongly inhibited by agents that collapse or prevent the formation of a transmembrane H+gradient, but is completely insensitive to the phosphoryl transition state analog, vanadate. In contrast, ATP-dependent accumulation of the glutathione conjugate of a chlorsulfuron analog, chlorimuron-ethyl, is incompletely inhibited by agents that dissipate the transmembrane H+ gradient but completely abolished by vanadate. In both cases, however, conjugation is essential for net uptake because neither of the unconjugated parent compounds are accumulated under energized or nonenergized conditions. That the attachment of glucose to two naturally occurring phenylpropanoids, p-hydroxycinnamic acid and p-hydroxybenzoic acid via aromatic hydroxyl groups, targets these compounds to the functional equivalent of the transporter responsible for chlorsulfuron-glucoside transport, confirms the general applicability of the H+ gradient dependence of glucoside uptake. It is concluded that H+gradient-dependent, vanadate-insensitive glucoside uptake is mediated by an H+ antiporter, whereas vanadate-sensitive glutathione conjugate uptake is mediated by an ATP-binding cassette transporter. In so doing, it is established that liquid chromatography-mass spectrometry affords a versatile high-sensitivity, high-fidelity technique for studies of the transport of complex organic molecules whose synthesis as radiolabeled derivatives is laborious and/or prohibitively expensive.

MATE Transporters Facilitate Vacuolar Uptake of Epicatechin 3′-O-Glucoside for Proanthocyanidin Biosynthesis inMedicago truncatulaandArabidopsis

“…Modification of flavonoids and other compounds by processes such as glycosylation and acylation is often a necessary prerequisite for their transport into the vacuole by MATE or ABC transporters (Klein et al, 1996;Bartholomew et al, 2002;Marinova et al, 2007;Gomez et al, 2009), and flavonoid aglycones are generally not suitable substrates for MATE transporters. Arabidopsis TT12 transports Cy3G but not C3G, flavonoid aglycones, or flavonol glucosides, such as quercetin-3-O-glucoside (Marinova et al, 2007).…”

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

“…Virtually all of the compound (.98%) accumulated in the vacuole as two Glc conjugates, a phenolic glucoside and a Glc ester, which both contained a single Glc molecule attached to the aromatic hydroxyl or carboxyl group of pHBA by a 1-O-b-D linkage. Glc conjugation, which takes place in the cytosol via distinct UDPglucosyltransferases , is required for vacuolar uptake, and recent studies have shown that the pHBA phenolic glucoside and Glc ester are transported by separate carriers that have different mechanisms (Bartholomew et al, 2002). The highest leaf content of pHBA Glc conjugates in nucleartransformed tobacco (Nicotiana tabacum) plants was 0.52% dry weight, which is equivalent to 0.24% free pHBA after correcting for the mass of the attached Glc residue.…”

Uptake and transformation of phenols by duckweed (Lemna gibba)

“…This implies that further research is still required to clarify their precise relationships because the glutathione conjugation of xeonobiotics is well known to be a prerequisite reaction for their efficient transfer to the vacuole as it is described with glycosylation. 27) The major secondary metabolite observed in duckweed exposed to I-VI was the glycoside conjugates. Glycoside conjugation is known to be one of the most common transformations for chemicals having the amino, hydroxy or carboxy group in plants.…”