Figure 3. Transgenic lines containing the vacuole-targeted SacB gene.

A. Fructan concentration was determined for seven individual seeds containing the SacB gene in a wild-type (626.1 and 250.1) and homozygous mutant background (sh2-626, shr4085, shr226, bt2-226 and sh,-626). Seeds were harvested at 45 - 50 DPP and dried, prior to analysis.

B. Homozygous mutant seeds segregated on the ear for the SacB gene. Dry weight analysis of mutants containing fructan (dark bars) was compared to mutant seeds that did not contain the SacB gene (light bars). Fructan was not detected in mature wild-type seeds. Redrawn from Caimi et al. Plant Physiol. 110: 355-363

(1996) with permission.

10 DPP, it is restricted to the basal region of the kernel where starch does not accumulate^3,34], It is also unlikely that an endogenous invertase would degrade polymer in the upper endosperm consistently to 1% of the seed weight without going beyond that level, resulting in complete hydrolysis. The results are consistent with a report of fructan accumulation in the vacuole of potato tubers [35]. A Bacillus subtilis SacB gene fused to vacuole targeting signals resulted in accumulation of polymer accounting for 1-7% of the dry weight of microtubers. Fructan accumulated in transgenic potato leaves and microtubers was shown to be stable, not subject to degradation by invertase. Degradation of polymer by native invertase activity can not, therefore, fully explain the low levels of polymer accumulated in vacuoles of transgenic plants.

In a relatively simple one enzyme-one substrate system, where gene expression does not appear to be limiting, it is very possible that substrate (sucrose) concentration in the vacuole is a significant factor limiting polymer synthesis. Manipulating sucrose concentration in maize kernels and determining the effect on polymer synthesis would be helpful in establishing the role of substrate limitation in polymer synthesis and can be accomplished by expressing the prokaryotic gene in one of many well-studied maize starch mutants. Maize mutants containing defects at various points in the starch biosynthetic pathway accumulate higher levels of sucrose at the expense of starch. The level of sucrose varies among the mutants. For example,

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