A Group Translocator for Sucrose Assimilation in Tonoplast Vesicles of Sugarcane Cells

Abstract

ABSTRACIExistence of a group translocator for sucrose transfer into vacuoles of sugarcane (Saccharum sp.) cells has been further confirmed by the use of tonoplast vesicles isolated from intact vacuoles. The group translocator depends on external UDP-Glucose (Glc) and, via a series of enzymic reactions within the tonoplast, sucrose phosphate and sucrose are deposited inside the vesicles. Fructose-6-phosphate was not required for UDPGic uptake, nor was it taken up. None of the other supr phosphates tested were taken up nor were the nucleotide suprs, UDP-Galactose and ADP-Glc. The uptake of UDP-GIc was concentration-dependent with a K,. of 1.2 millimolar and a V,,, of 83.3 nanomoles per minute per milligram protein. The optimum pH for UDP-Glc uptake was 7.0. Uptake of UDP-Glc was inhibited by para-chloromercuribenzene-sulfonic acid, UDP, and GDP;, carbonyl cyanide m-chlorophenylhydrazone inhibited to a lesser extent.A protonmotive force across membranes of higher plant cells that energizes the transport of molecules against a concentration gradient is now widely known and documented (5). The transport of sugars can proceed not only across the plasmalemma but also across the tonoplast (5). Sugarcane cells grown in suspension cultures utilize a proton-antiport mechanism to accumulate glucose across the tonoplast (9), but the rate of sucrose uptake by this mechanism is marginal and cannot account for the rate at which sucrose appears inside the vacuole (8). While exogenously added MgATP stimulates sucrose uptake into red beet vacuoles (2), a similar stimulation in sugarcane vacuoles has not been observed.These findings led us to consider an alternative mechanism by which sucrose might accumulate in vacuoles, and a group translocation mechanism for the accumulation of sucrose across the tonoplast has been identified in intact vacuoles isolated from sugarcane cell suspension cultures (10). By this process, sucrose formation is dependent on availability of UDP-Glc but independent of Fru-6-P on the cytoplasmic side of the vacuole. A stoichiometry of two molecules of UDP-Glc consumed for each Suc2-P or sucrose molecule formed was deduced from the data, UDP-Glc serving both as the direct substrate for Suc-P synthase and as a source of Fru-6-P. To conform to the observations made with vacuoles in bringing about the conversion of UDP-Glc to ' Published with the approval of the Director as Paper No. 597 free sucrose, several enzymes must be closely associated with an enzyme complex within or on the tonoplast. The vectorial nature of the reaction sequence is borne out by distribution of intermediates and products inside the vacuole and in the incubation medium.Intact vacuoles provided a useful system with which initially to show the uptake of UDP-Glc and its conversion to sucrose (10). The evidence for the proposed group translocator could be substantially strengthened if its functions were demonstrated in tightly sealed tonoplast vesicle preparations purged of solutes normally associated with the vacuole. Furthermore, chara...

Evidence for the Uptake of Sucrose Intact into Sugarcane Internodes

“…Maretzki and Thom (16,20) have presented the strongest evidence that sucrose must be cleaved and resynthesized for transport into sugarcane, although the presence of a group transporter has recently been questioned (17,18). The data presented here also would seem to be in conflict with their initial data.…”

“…However, the ratio of labeled glucose to fructose in sucrose in the storage compartment was low in all tissues, indicating less than 15% randomization of label. The group transporter hypothesis of Maretzki and Thom (16,20) requires complete randomization for uptake into the vacuole, since they have reported that ['4C]fructose was not taken up by isolated vacuoles, but required conversion through UDPG for transport. In this study, however, although some randomization of label was observed in sucrose from ethanol extracts, it was not nearly enough to substantiate a requirement for sucrose cleavage and resynthesis during transport.…”

“…(10)(11)(12). Maretzki and Thom (16,20) proposed a group transporter functioning at the tonoplast to be responsible for sucrose transport in sugarcane, but recent reports have suggested that the earlier results may have been in error (17,18). A group translocator for sucrose uptake has also been proposed for beet roots (9,19) and grape berries (5).…”

Sucrose Phosphatase Associated with Vacuole Preparations from Red Beet, Sugar Beet, and Immature Sugarcane Stem

“…The presence of the specific enzyme in plants probably indicates its importance in sucrose synthesis (9), but it could also be involved in the transport of sucrose, particularly in sugarcane and red beet storage tissues (14,18,19), where a group translocator has been postulated to operate. Our results showing the enzyme to be present in vacuole preparations from storage tissues support the view that sucrose phosphatase could participate in their sucrose transport.…”

“…Sucrose, maltose, melezitose, turanose, Pi, PPi, and F-cause inhibition of activity. Sucrose phosphate had been suggested as an intermediate in sucrose transport across the tonoplast in some plants (3,6,8) and recent evidence strongly supports the involvement of sucrose phosphate in a group translocation mechanism for sucrose transfer into isolated vacuoles of both sugarcane and red beet and into tonoplast vesicles of sugarcane (14,18,19). UDPGlucose was the external substrate for the group translocator and sucrose and sucrose phosphate with or without added MgATP were not taken up by the sugarcane vacuoles or vesicles (14,18).…”