Fructan Accumulation and Sucrose Metabolism in Transgenic Maize Endosperm Expressing a Bacillus amyloliquefaciens SacB Gene

Abstract

Over 40,000 species of plants accumulate fructan, p-2-1-and P-2-6-linked polymers of fructose as a storage reserve. Due to their high fructose content, severa1 commercial applications for fructans have been proposed. However, plants that accumulate these polymers are not agronomically suited for large-scale cultivation or processing. This study describes the transformation of a Bacillus amyloliguefaciens SacB gene into maize (Zea mays 1.) callus by particle bombardment. Tissue-specific expression and targeting of the SacB protein to endosperm vacuoles resulted in stable accumulation of high-molecular-weight fructan in mature seeds. Accumulation of fructan in the vacuole had no detectable effect on kernel development or germination. Fructan levels were found to be approximately 9-fold higher in sh, mutants compared to wild-type maize kernels. In contrast to vacuole-targeted expression, starch synthesis and endosperm development in mature seeds containing a cytosolically expressed SacB gene were severely affected. The data demonstrate that hexose resulting from cytosolic SacB activity was not utilized for starch synthesis. Transgenic seeds containing a chimeric SacB gene provide further evidence that the dominant pathway for starch synthesis in maize endosperm is through uridine diphosphoglucose catalyzed by the enzyme sucrose synthase.

Cytosolic expression of the Bacillus amyloliquefaciens SacB protein inhibits tissue development in transgenic tobacco and potato

“…Invertase activity was defined as the amount of free fructose formed from sucrose. Fructose levels were determined by subtracting the amount of total hexose formed by the amount of free glucose formed in separate enzyme linked assays, described previously (Caimi et al, 1996). Hexose, sucrose and larger fructose oligomers present following incubation with sucrose were also analysed by high-performance anion-exchange chromatography, described below.…”

“…The cassette also contained a 503 bp 3' end from the maize chemically induced 2-1 gene (Hershey & Stoner, 1991). The bacterial secretion signal was removed from the SacB gene and unique restriction enzyme sites were added by PCR, previously described in Caimi et al (1996). Restriction enzyme sites were also added by linker ligation or PCR to the promoter region.…”

“…Plants containing a fructosyltransferase fused to vacuole-targeting signals accumulate fructan {c. 3-8 % of tissue d. wt) with no discernible effect on plant development . However, accumulation of similar levels of fructan in the cytosol of maize endosperm resulted in an extreme detrimental phenotype (Caimi et al, 1996).…”

High level accumulation of α-glucan in maize kernels by expressing the gtfD gene from Streptococcus mutans

“…However, we failed to observe any obvious alteration in glucan accumulation in the gtfD sh2 background, suggesting that either the elevated level of sucrose was not routed to the amyloplast where GTF-D presumably functioned or sucrose level was not the limiting factor for a higher level of glucan production. Caimi et al (1996) reported that a dramatic increase of fructan accumulation in the sh2 mutant when targeting a SacB gene in the maize endosperm vacuoles. There is no doubt that the vacuolar sucrose level is higher in the sh2 background as compared with that in the cognate wildtype lines.…”

“…Fructan is also synthesized from sucrose via a single enzymatic step catalyzed by fructosyltransferases whose reaction mechanisms are similar to GTFs (Chambert and GonzyTreboul 1976). Fructan biosynthesis was reported to be successfully induced in potato, sugar beet, maize, tobacco, and chicory by ectopic expression of fructosyltransferases from plant or bacterial sources Caimi et al 1996;Rober et al 1996;Sevenier et al 1998;Sprenger et al 1997). Ebskamp et al (1994) introduced the SacB gene from B. subtilis into tobacco, resulting in 3-8% fructan accumulation in the tobacco leaves; Caimi et al (1996) transformed maize with the SacB gene from B. amyloliquefaciens, leading to fructan accumulation up to 4-8% of the dry weight of mature seeds.…”