E

Figure 5. Fructan concentration and dry weight was determined for 54 seeds containing the vacuole-targeted SacB gene. Seeds were harvested at 48 DPP and dried, prior to analysis (R2 = 9E 06). Caimi, unpublished data.

Seed Weight (mg)

Figure 5. Fructan concentration and dry weight was determined for 54 seeds containing the vacuole-targeted SacB gene. Seeds were harvested at 48 DPP and dried, prior to analysis (R2 = 9E 06). Caimi, unpublished data.

2.2 Additional Considerations

Future work leading to higher levels of fructan accumulation in the vacuole of transgenic maize endosperm will undoubtedly be tied to ongoing research in native fructan-producing plants. Understanding the fate of sucrose in a cell is an important area of plant research. Determining the nature of how plants redirect the flow of sucrose into alternate forms of carbohydrate is particularly important in fructan-producing species. Diverting the flow of sucrose in transgenic plants represents a valuable tool in describing not only the role of enzymes in specific metabolic pathways, but also the contribution of carrier-mediated transport in partitioning sucrose within a cell.

Active sucrose transport may be involved in fructan synthesis in the leaves of many temperate grasses [7]. Preventing sucrose export from leaves by excision has proven to be an excellent model for rapid induction of fructan synthesis [41-43]. Fructan accumulates in excised leaves of Lolium temulentum after only a short 8-hour lag-time [41]. The rate of synthesis was estimated to be approximately 1.8 mg g"1 If1 [44]. Polymer synthesis in excised wheat leaves was evident after less than 5 hours [42], The rate of polymer synthesis in grass leaves has led to the suggestion that a carrier-mediated tonoplast transport mechanism is present in fructan-producing plants [7], Passive transport of sucrose across the tonoplast would be too slow and unresponsive to the rapid synthesis of polymer.

It is not known for certain whether active sucrose transport into vacuoles is crucial to synthesis in all fructan-producing species or, for that matter, in transgenic plants containing a chimeric fructosyltransferase gene. Sucrose transport into sugar beet vacuoles was reported to be by an active, carrier-mediated, mechanism [45]. Recently, Sevenier et al. [46] reported very high levels of fructan accumulation in transgenic sugar beets containing a chimeric Jerusalem artichoke SST gene, accounting for more than 40% of the taproot dry weight. However, the

Jerusalem artichoke SST gene expressed in transgenic petunia resulted in far less accumulation [21]. Active transport of sucrose into the vacuoles of sugar beet taproots and passive transport in petunia could explain the difference in fructan accumulated in the two plants expressing the same gene. The results may also suggest that tonoplast transporters mediate partitioning of sucrose in many, if not all fructan-producing plants.

The potential role of active transport in partitioning sucrose to vacuoles could be clarified by altering expression of a tonoplast sucrose transporter gene in transgenic plants. Numerous sucrose transporters, localized to the plasma membrane, have been isolated [47], It is curious, however, that tonoplast sucrose transporters have not yet been recovered. One possible explanation for this is that tonoplast transporters function differently and are not related to the known sucrose-proton symporter gene family. Evidence showing that the two types of transporters function differently and are not related may be seen by comparing sucrose transport into barley vacuoles (a fructan-producing plant) to that of an isolated spinach sucrose-proton symporter. The pH optimum for activity was shown to be very different for the two transport systems and a 10-fold difference in affinity for sucrose was reported [49-50], A threshold level of sucrose was also shown to be necessary prior to fructan synthesis in barley [9], Creating a sucrose gradient by fructan synthesis in leaves is believed to facilitate the flow of sucrose into the vacuole [44]. The results demonstrate that export of sucrose from the cell is the preferred route and when this is prevented, the flow of sucrose is then diverted to the vacuole. Sucrose-proton symporters are not known to be subject to activation by a gradient. They act by accumulating substrate against a steep gradient. The effect of accumulating sucrose, creating a gradient prior to transport, is reminiscent of porin-like activity. A porin that facilitates sucrose transport has been demonstrated in bacteria where the rate of transport is proportional to substrate concentration [51-52].

Collectively, the data strongly suggests that a carrier mediates sucrose transport into the vacuole of fructan-producing plants (whether by a porin or unusual proton-symporter) and that creating a sucrose gradient alone in transgenic plants is not sufficient to support high levels of fructan synthesis. Identification and characterization of tonoplast sucrose transporters in the future will undoubtedly have a significant impact on the level of fructan produced in vacuoles of transgenic starch-storing crops.

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