The ABC-like vacuolar transporter for rye mesophyll flavone glucuronides is not species-specific.

In many cases, the vacuolar uptake of secondary metabolites has been demonstrated to be strictly specific for a given compound and plant species. While most plants contain glycosylated secondary substances, few cases are known where flavonoids may also carry negative charges, e.g. as glucuronide conjugates. Vacuolar transport of glucosylated phenylpropanoid derivatives has been shown to occur by proton substrate antiport mechanisms (Klein, M., Weissenböck. G., Dufaud, A., Gaillard, C., Kreuz, K., Martinoia, E., 1996. Different energization mechanisms drive the vacuolar uptake of a flavonoid glucoside and a herbicide glucoside. J. Biol. Chem. 271, 29,666-29,671). In contrast, flavone glucuronides appearing specifically in rye mesophyll vacuoles are taken up by direct energisation utilising MgATP, strongly arguing for the presence of an ATP-binding cassette (ABC) transporter belonging to the subfamily of multidrug resistance-associated proteins (MRP) on the rye vacuolar membrane (Klein, M., Martinoia, E., Hoffmann-Thoma, G., Weissenböck, G., 2000. A membrane-potential dependent, ubiquitous ABC-like transporter mediates the vacuolar uptake of rye flavone glucuronides regulation of glucturonide uptake by glutathione and its conjugates. Plant Journal 21, 289-304). MRPs are known to transport negatively charged organic anions. Results presented here suggest that the vacuolar directly energised MRP-like glucuronate pump for plant-specific flavone glucuronides is ubiquitously present in diverse plant species since rye flavone glucuronides are taken up into vacuoles isolated from the barley mesophyll or from the broccoli stalk parenchyma representing two species which do not synthesise glucuronidated secondary compounds. According to the transport characteristics and inhibition profile observed we propose the existence of a high-capacity, uncoupler-insensitive vacuolar ABC transporter for flavone glucuronides and possibly other negatively charged organic compounds -- plant-born or xenobiotic -- irrespective of the plant's capability to endogenously produce glucuronidated compounds.

Ultrastructure of minor-vein phloem and assimilate export in summer and winter leaves of the symplasmically loading evergreens Ajuga reptans L., Aucuba japonica Thunb., and Hedera helix L.

Minor-vein ultrastructure and sugar export were studied in mature summer and winter leaves of the three broadleaf-evergreen species Ajuga reptans var. artropurpurescens L., Aucuba japonica Thunb. and Hedera helix L. to assess temperature effects on phloem loading. Leaves of the perennial herb Ajuga exported substantial amounts of assimilates in form of raffinose-family oligosaccharides (RFOs). Its minor-vein companion cells represent typical intermediary cells (ICs), with numerous small vacuoles and abundant plasmodesmal connectivity to the bundle sheath. The woody plants Hedera and Aucuba translocated sucrose as the dominant sugar species, and only traces of RFOs. Their minor-vein phloem possessed a layer of highly vacuolated cells (VCs) intervening between mesophyll and sieve elements. Depending on their location and ontogeny, VCs were classified either as companion or parenchyma cells. Both cell types showed symplasmic continuity to the adjacent mesophyll tissue although at a lower plasmodesmal frequency compared to the Ajuga ICs. p-Chloromercuribenzenesulfonic acid did not reduce leaf sugar export in any of the plants, indicating a symplasmic mode of phloem loading. Winter leaves did not show symptoms of frost injury, and the vacuolar pattern in ICs and VCs was equally prominent in both seasons. Starch accumulation as a result of reduced phloem loading was not observed to be triggered by low temperature. In contrast, high amounts of starch were found in mesophyll and bundle-sheath cells of summer leaves. Physiological data on season-dependent leaf exudation showed the maintenance of sugar export in cold-acclimated winter leaves.

A membrane-potential dependent ABC-like transporter mediates the vacuolar uptake of rye flavone glucuronides: regulation of glucuronide uptake by glutathione and its conjugates.

In this paper we present results on the vacuolar uptake mechanism for two flavone glucuronides present in rye mesophyll vacuoles. In contrast to barley flavone glucosides (Klein et al. (1996) J. Biol. Chem. 271, 29666-29671), the flavones luteolin 7-O-diglucuronyl-4'-O-glucuronide (R1) and luteolin 7-O-diglucuronide (R2) were taken up into vacuoles isolated from rye via a directly energized mechanism. Kinetic studies suggested that the vacuolar glucuronide transport system is constitutively expressed throughout rye primary leaf development. Competition experiments argued for the existence of a plant MRP-like transporter for plant-specific and non-plant glucuronides such as beta-estradiol 17-(beta-D-glucuronide) (E217G). The interaction of ATP-dependent vacuolar glucuronide uptake with glutathione and its conjugates turned out to be complex: R1 transport was stimulated by dinitrobenzene-GS and reduced glutathione but was inhibited by oxidized glutathione in a concentration-dependent manner. In contrast, R2 uptake was not increased in the presence of reduced glutathione. Thus, the transport system for plant-derived glucuronides differed from the characteristic stimulation of vacuolar E217G uptake by glutathione conjugates but not by reduced glutathione (Klein et al. (1998) J. Biol. Chem. 273, 262-270). Using tonoplast vesicles isolated with an artificial K+ gradient, we demonstrate for the first time for plant MRPs that the ATP-dependent uptake of R1 is membrane-potential dependent. We discuss the kinetic capacity of the ABC-type glucuronide transporter to explain net vacuolar flavone glucuronide accumulation in planta during rye primary leaf development and the possibility of an interaction of potential substrates at both the substrate binding and allosteric sites of the MRP transporter regulating the activity towards a certain substrate.