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

Abstract

SUMMARYFructans are linear or brancbed polymers containing a single sucrose and repeating fructose residues. An early model for fructan biosyntbesis in bigber plants suggested that partial synthesis of tbe polymer occurred in tbe cell cytosol. Tbe current model suggests tbat synthesis requires the interaction of two separate fructosyltransferases located in the vacuole. Tobacco lines containing a chemically induced promoter, directing expression of the Bacillus amyloliquefaciens SacB gene in tbe present study, provided an opportunity to regulate and target fructan synthesis to tbe cytosol of transgenic plants. Induced expression of tbe gene led to rapid destruction of leaf tissue. Amino acid substitution at a bigbly conserved site (Arg^^^) in tbe SacB gene reduced tbe fructosyltransferase efficiency witbout reducing tbe invertase activity of tbe enzyme. Expression of tbe mutant gene in transgenic tobacco also resulted in leaf damage. Hov^^ever, the appearance of necrotic tissue was greatly delayed. The results suggest tbat tbe pbenotype is due to accumulation of fructan in tbe cytosol. Fructan metabolism in tbe cytosol of potato tubers was also detrimental to development. Tuber size and starcb synthesis was significantly reduced in lines containing tbe untargeted gene. Transgenic tobacco and potato containing tbe SacB gene offer an opportunity to study tbe metabolism of fructan and the effect of accumulation on plant cell development.

Transgenic potato ( Solanum tuberosum ) tubers synthesize the full spectrum of inulin molecules naturally occurring in globe artichoke ( Cynara scolymus ) roots

“…In contrast to results obtained for expression of bacterial fructosyltransferases in plants (27,32), we observed neither adverse phenotypic effects nor a reduction of tuber yield for inulin-accumulating potato plants. Furthermore, the impact on carbohydrate metabolism of green plant organs was only minor and leaf inulin content was low.…”

Dissecting the roles of osmolyte accumulation during stress

“…1). None the less, in contrast to fructan-accumulating transgenic lines which have higher levels of non-structural carbohydrates (Pilon-Smits et al 1995;Caimi et al 1997;Turk et al 1997), transgenic trehalose- (Romero et al 1997) and mannitol-accumulating plants have lower levels of sucrose (Tarczynski et al 1992;Thomas et al 1995). Whether the slight decreases observed in mtlD transformants might have a significant impact is doubtful (Thomas et al 1995;Karakas et al 1997).…”

“…1, the likely involvement of activated flux through hexokinase complicates elucidation of the molecular basis of stress tolerance arising from increases in trehalose and fructan synthesis (Table 1). Phenotypes which have been observed in various trehalose-and fructan-accumulating transgenic lines, including stunted growth (Holmström et al 1996;Caimi et al 1997;Goddijn et al 1997;Romero et al 1997;Turk et al 1997), chlorotic and necrotic leaves (Goddijn et al 1997;Romero et al 1997;Caimi et al 1997;Turk et al 1997) and a reduction in apical dominance (Goddijn et al 1997;Romero et al 1997), are reminiscent of those typically associated with elevated sugar levels (Jang et al 1997).…”

High levan accumulation in transgenic tobacco plants expressing the Gluconacetobacter diazotrophicus levansucrase gene

“…Several groups have reported the transfer of bacterial fructosyltransferase genes into crops that do not naturally produce fructans, like tobacco Pilon-Smits et al, 1995;Caimi et al, 1997;Turk et al, 1997;Park et al, 1999;Gerrits et al, 2001;Konstantinova et al, 2002), potato PilonSmits et al, 1996;Caimi et al, 1997;Gerrits et al, 2001), maize (Caimi et al, 1996), sugar beet (Pilon-Smits et al, 1999), white clover (Jenkins et al, 2002) and poplar (Li et al, 2008). Relative The extracts from leaf, stem, root, flower, and seeds of three-month old plants were precipitated with 60% ethanol to concentrate the polyfructan.…”