Figure 4. Fructan concentration was determined from ten seeds, containing a vacuole-targeted SacB gene, collected at each time-point. Activity of the 10 kD zein promoter is represented by a dark line extending from approximately 12 DPP through 38 DPP. Peak promoter activity normally occurs at 19-24 DPP. Caimi, unpublished data.

the sugary-1 (sul) mutant results in a 2-fold increase in sucrose concentration over wild-type [36-37]. A disabled ADP-glucose pyrophosphorylase in the shrunken-2 (sh2) mutant results in even lower levels of starch and a 4-fold increase in sucrose concentration over wild-type maize [38-40]. Starch mutations are recessive therefore the transgenic lines must be crossed twice with the mutant, resulting in kernels containing the transgene in a homozygous mutant and wild-type background. Wild-type seeds containing the SacB gene serve as an internal control. If sucrose levels limit polymer synthesis in wild-type seeds, fructan accumulation would be expected to be higher in a homozygous mutant kernel. Figure 3A demonstrates that the level of polymer accumulated in every homozygous starch mutant background tested was significantly higher than in the wild-type controls. The highest level of polymer was found in the sh2 background, which also accumulates the highest level of sucrose. The results suggest that the concentration of sucrose in the vacuole of wild-type endosperm does indeed limit polymer synthesis.

Polymer accumulation accounted for slightly more than 8% of the dry weight in homozygous sh2 seeds. Thus, expression of the SacB gene provides a sink for sucrose in the mutant kernels. However, this newly acquired pathway did not result in increased dry matter accumulation. The dry weight of homozygous mutant kernels containing up to 8% fructan was no different than mutant seeds not containing the SacB gene (Figure 3B). The results indicate that although increased sucrose levels in the mutant seed led to higher levels of polymer, the new pathway does not act as a supplemental sink. The sequestration of carbon into fructan does not draw additional carbohydrate into a seed even though the starch pathway is not fully functional. Increased sink strength due to the additional metabolic activity should lead to increased dry matter accumulation (increased seed mass). This result suggests that the vacuole-targeted enzyme does not have unhindered access to sucrose. Additional evidence to suggest that the supply of sucrose does not adequately meet the capacity of a vacuole-targeted enzyme is seen in Figure 4. The 10 kD zein promoter is active early in endosperm development and peaks at approximately 19-24 DPP. This suggests that the majority of fructan should accumulate early in endosperm development. However, polymer accumulation was delayed and accumulation occurred slowly over approximately 40 DPP. The pattern of accumulation does not indicate that the enzyme has free access to substrate. In addition, no correlation could be established between polymer concentration and mature seed dry weight in the sh2 mutant background (Figure 5). This was true for every mutant (shrunkenl, sugaryl, brittle2) tested (Caimi, unpublished observations). Accumulating water-soluble polysaccharide in the seed, therefore, does not negatively affect the final mutant seed weight. If polymer does not cause a reduction in mutant seed weight, higher levels of accumulation may be possible, assuming that sucrose availability is not limited.

The data from transgenic maize, containing the vacuole-targeted SacB gene demonstrates that (i) low levels of fructan are accumulated in seeds, (ii) synthesis of polymer occurs later in development than expected and (iii) expression of the enzyme does not add to the sink capacity of seeds. Collectively, the data are consistent with the suggestion that sucrose enters the vacuole in endosperm by a passive process. This is also the proposed mechanism of sucrose transport into vacuoles of tobacco, spinach and maize leaves [41]. Diffusion across the tonoplast is a slow process and although the SacB enzyme converts available substrate into polymer in transgenic seeds, replenishment with additional sucrose, resulting in higher levels of fructan and increased seed mass, would not be expected.

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