Wt 6261 6251 626132 62522 4085 2501 25073

Figure 2. Fructan concentration for each independent line was determined from seven individual seeds (dark bars) containing the vacuole-targeted SacB gene. Polymer was not detected in mature wild-type (WT) seeds. Seeds were harvested at 45 - 50 DPP and dried, prior to analysis (dry weight, dw). Only the transgenic line 250.73 was homozygous for SacB. The number of intact SacB gene copies varied among independent transformed lines. Redrawn from Caimi et al. Plant Physiol. 110: 355-363 (1996) with permission.

Although the large bacterial-like polymer was produced in transgenic starch-storing tissue, the level of accumulation was not very high. Fructan accounted for only 1% of the mature seed dry weight in several independent transformed lines (Figure 2). This was true for heterozygous (In) as well as homozygous plants containing a minimum of three endosperm copies of the SacB gene (3n). Delivery of the chimeric gene to embryogenic maize callus was by particle bombardment. Control over integration of intact gene copy number by this method is at best, difficult. Therefore, independent transgenic lines often contain varying numbers of intact genes [30]. Comparing transgenic maize lines containing few intact SacB copies to those with a much more complex pattern of integration revealed that there was no relationship between gene copy number and polymer accumulation [29]. Transgenic lines with multiple intact SacB copies did not accumulate higher levels of polymer than lines with fewer copies.

The results strongly suggest that gene expression does not influence the level of polymer produced in seeds. This naturally leads to the question of what are the factor(s), limiting polymer accumulation in vacuoles of maize endosperm?

Expression of the SacB gene was directed by a 10 kD zein promoter which is known to be active early, 10-12 days post-pollination (DPP), in endosperm development [31]. Activity of the promoter remains high through at least 30 DPP. Continuous expression of the SacB gene should lead to polymer synthesis through this period of seed development. It is possible that only low levels of polymer are synthesized because the bacterial enzyme must compete for sucrose with an endogenous vacuolar invertase. Alternatively, vacuolar invertases could degrade polymer creating a futile cycle of synthesis and degradation. However, sucrose metabolism in the upper endosperm of maize is mediated by sucrose synthase, not invertase [32]. Invertase activity is down-regulated very early in development and although present after

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