Abstract
Until now, specific inhibitors of sucrose carriers were not available. This led us to study the properties of the recently synthesized D-glucose-fenpiclonil conjugate (D-GFC). This large amphiphilic glucoside exhibited an extremely low phloem systemicity in contrast to L-amino acid-fenpiclonil conjugates. Using Ricinus seedlings, the effect of D-GFC on 0.5 mM [14C]sucrose (Suc), 3-O-[3H]methylglucose, and [3H]glutamine uptake by cotyledon tissues was compared with that of p-chloromercuribenzenesulfonic acid (PCMBS). D-GFC dramatically inhibited H+-Suc symport at the same concentrations as PCMBS (0.5 and 1 mM), but in contrast to the thiol reagent, it did not affect 3-O-methylglucose and glutamine transport, nor the acidification of the incubation medium by cotyledon tissues. Similarly, 0.5 mM D-GFC inhibited active Suc uptake by Vicia faba leaf tissues and by Saccharomyces cerevisiae cells transformed with AtSUC2, a gene involved in Suc phloem loading in Arabidopsis, by approximately 80%. The data indicated that D-GFC was a potent inhibitor of Suc uptake from the endosperm and of Suc phloem loading. It is the first chemical known to exhibit such specificity, at least in Ricinus, and this property permitted the quantification of the two routes involved in phloem loading of endogenous sugars after endosperm removal.
Highlights
The non-permeant or poorly permeant sulfhydryl reagent p-chloromercuribenzenesulfonic acid (PCMBS) has been successfully used in phloem biology, first to demonstrate that sucrose accumulates in the phloem symplasm from the vein apoplasm through Suc carriers as in Beta vulgaris and Vicia faba (Giaquinta, 1976; Delrot et al, 1980; Giaquinta, 1983), and to identify apoplasmic and symplasmic loaders (van Bel et al, 1992; Turgeon and Medville, 2004; Turgeon and Ayre, 2005)
PCMBS dramatically inhibits the activity of Suc carriers but because it reacts with cysteine residues of many other plasma membrane (PM) intrinsic proteins, it affects the transport of other solutes, as demonstrated in Abbreviations: CF, concentration factor; Gln, glutamine; D-GFC, D-glucose–fenpiclonil conjugate; 3-O-MeG, 3-O-methylglucose; PCMBS, p-chloromercuribenzenesulfonic acid sodium salt; PM, plasma membrane; Suc, sucrose
We have previously described the detailed synthesis of the D-glucose– fenpiclonil conjugate (D-GFC; Fig. 1) (Wu et al, 2016)
Summary
The non-permeant or poorly permeant sulfhydryl reagent p-chloromercuribenzenesulfonic acid (PCMBS) has been successfully used in phloem biology, first to demonstrate that sucrose accumulates in the phloem symplasm from the vein apoplasm through Suc carriers as in Beta vulgaris and Vicia faba (Giaquinta, 1976; Delrot et al, 1980; Giaquinta, 1983), and to identify apoplasmic and symplasmic loaders (van Bel et al, 1992; Turgeon and Medville, 2004; Turgeon and Ayre, 2005). The effect of PCMBS on the PM H+-ATPase varies with tissues It does not significantly affect the proton pumping activity nor the transmembrane potential difference in mature broad bean leaf tissues (Delrot et al, 1980; Bourquin et al, 1990), but it inhibits proton pumping by microsomal vesicles and acidification of the incubation medium by intact cotyledons of Ricinus communis seedlings (Williams and Hall, 1987; Williams et al, 1990). As in most plant species (Liu et al, 2012), sucrose (Suc) is the major sugar of the Ricinus phloem sap, with concentrations of approximately 300 mM in intact seedlings (Kallarackal et al, 1989; Verscht et al, 1998). When cotyledons are dipped in solutions containing from 25 to 200 mM glucose, the same concentrations as in the incubation solutions are found in the phloem sap after 2 h of incubation (Kallarackal and Komor, 1989)
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