SummaryIn this paper we present results on the vacuolar uptake mechanism for two¯avone glucuronides present in rye mesophyll vacuoles. In contrast to barley¯avone glucosides (Klein et al. (1996) J. Biol. Chem. 271, 29666±29671), the¯avones 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 energised 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-speci®c and non-plant glucuronides such as b-estradiol 17-(b-D-glucuronide) (E 2 17G). 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 E 2 17G 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 arti®cial K + gradient, we demonstrate for the ®rst 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¯avone 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.

J, 1996, Sucrose accumulaeion in sweet sorghum stem internodes in relation tO ' growth, Sweet sorghum (Sorghum bicoior L, Moench) stems of different cultivars (NK 405, Keller and Tracy) reveal a different pattem of sucrose accumn]ation with respect to intemodal sugar content and distribution. The onset of sucrose storage is not necessarily associated with the reproductive stage of the plant, as was hitherto assumed, but obviously occurs after cessation of internodal elongation as was postulated for the sugarcane stem. For at least two of the three cultivars, ripening is an internode to internode process beginning at the lowermost culm parts. Intensive growth of the intemodes, combined with a high hexose content in stem parenchyma, shows a strong positive correlation (r > 0,94') to the activity of sucrose synthase (SaSy; EC 2,4,13), but not !0 invertase (EC 3,2,1,26) which is not present as soluble (neutral .and acid) or cell wallbound, salt-extractable enzyme in the three cultivars investigated. Sucrose synthase measured in sucrose cleavage and synthesis direction reveals divergent activity rates and sensitivity towards exogenously applied Mg"* ions and pH, SuSy activity' is connected to the increase of internodal sucrose content in so far as (I) its decline is a prerequisite for the onset of sucrose accumulation and (2) it remains at a constant low level during sucrose storage. Sucrose phosphate synthase (SPS; EC 2,4,1.14) activity in the sorghum stem is low compared to SuSy and uniformly distributed over all internodes. Only source leaves of sorghum show a considerable SPS activity, but neither stem nor leaf SPS reveal a positive correlation to the increase of internodal sucrose content. Sucrose phosphate phosphatase (SPP; EC 3.1.3,24) amounts to only 24-30% of the respective SPS activity bul follows the same distribution pattern. None of the enzymes under study proves to be responsible for the extent of sucrose storage in the stem, so other phenomena such as transport processes within the stem tissue require further investigation.

No abstract

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.

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 Given the fact that plant species with a type 1 (symplasmic) minor vein ultrastructure seem to dominate in the tropics and subtropics, and species with a type 2 (apoplasmic) minor vein ultrastructure in the temperate and boreal climate zones, a cold sensitivity of symplasmic phloem loading was postulated. Electron microscopic observations were taken as support for this proposal. The objective of the present work was to test this postulate by measuring physiological parameters correlated to phloem loading. Carbohydrate levels in the leaf, minor vein loading of 14CO2-derived assimilates in leaf segments and exudation of sugars and 14C-labelled compounds in several species from families with known phloem-loading pathways were compared in 10 and 20˚C-adapted plants at both 10 and 20˚C. No essential differences in response to temperature between symplasmically and apoplasmically phloem-loading species were observed. Carbohydrate availability for export was essentially similar, phloem loading was fully operative at 10˚C, and exudation of sugars equally reacted to low temperature in symplasmic and apoplasmic species. Apparently, the geographical distribution of type 1 and 2 species is not explained by a difference in temperature sensitivity of the phloem-loading mode.