Fructan as a New Carbohydrate Sink in Transgenic Potato Plants.

Abstract

Fructans are polyfructose molecules that function as nonstructural storage carbohydrates in several plant species that are important crops. We have been studying plants for their ability to synthesize and degrade fructans to determine if this ability is advantageous. We have also been analyzing the ability to synthesize fructan in relation to other nonstructural carbohydrate storage forms like starch. To study this, we induced fructan accumulation in normally non-fructan-storing plants and analyzed the metabolic and physiological properties of such plants. The normally non-fructan-storing potato plant was modified by introducing the microbial fructosyltransferase genes so that it could accumulate fructans. Constructs were created so that the fructosyltransferase genes of either Bacillus subtilis (sacB) or Streptococcus mutans (ftf) were fused to the vacuolar targeting sequence of the yeast carboxypeptidase Y (cpy) gene. These constructs were placed under the control of the constitutive cauliflower mosaic virus 35S promoter and introduced into potato tissue. The regenerated potato plants accumulated high molecular mass (>5 [times] 106 D) fructan molecules in which the degree of polymerization of fructose units exceeded 25,000. Fructan accumulation was detected in every plant tissue tested. The fructan content in the transgenic potato plants tested varied between 1 and 30% of dry weight in leaves and 1 and 7% of dry weight in microtubers. Total nonstructural neutral carbohydrate content in leaves of soil-grown plants increased dramatically from 7% in the wild type to 35% in transgenic plants. Our results demonstrated that potato plants can be manipulated to store a foreign carbohydrate by introducing bacterial fructosyltransferase genes. This modification affected photosynthate partitioning in microtubers and leaves and increased nonstructural carbohydrate content in leaves.

Improved Performance of Transgenic Fructan-Accumulating Tobacco under Drought Stress

“…Also these fructans are recognized by a monoclonal antibody directed against the P-2-6-linkage type (our unpublished results), which is not found in dicotyledons that accumulate fr xtans. The presence of the bacterial gene in these plants was established by Southern blotting, but no mRNA could be detected in transgenic tobacco or potato, in spitct of the 35s promoter used and in spite of the high fructan levels obtained in some of these plants (Ebskamp et a1 , 1994;Van der Meer et al, 1994). Fructan biosynthetic enzyme activity can be detected in these transgenic species tlut only by using a sensitive assay (Van der Meer et al, 1994, and our unpublished results).…”

“…Since no plant genes involved in fructan metabolism have yet been isolated, the SacB gene from Bacillus subtilis was used, encoding levansucrase (Steinmetz et al, 1985). Transgenic plants have been produced that accumulate bacterial fructans using this construct Van der Meer et al, 1994). The isogenic tobacco (Nicotiana tabacum) lines differing in the presence of fructans only provide ideal material to investigate the effect of fructans on growth performance and stress resistance.…”

Effects of drought, transgenic expression of a fructan synthesizing enzyme and of mycorrhizal symbiosis on growth and soluble carbohydrate pools in tobacco plants

“…This has been accomplished by expressing a bacterial gene encoding levansucrase Van der Meer et al, 1994 ;Pilon-Smits et al, 1995 ;Ro$ ber et al, 1996). Recently, a cDNA encoding a fructan-synthesizing enzyme has also been isolated from a plant source, namely sucrose : fructan 6-fructosyl transferase (6-SFT) from barley (Sprenger et al, 1995).…”

The vacuolar sorting domain of sporamin transports GUS, but not levansucrase, to the plant vacuole

“…This extraction method was repeated once with 300 //I of sterile water at 90 °C, once with 300 /*1 of 80 % ethanol at room temperature and once with 100/tl of sterile water at room temperature, and the supernatants were combined. The pellet was used for starch determination as described by Van der Meer et al (1994). Eructan content of the leaf material was determined by spotting 1 //I of the supernatant on silica gel TLC foils (Schleicher & Schuell) as described by Cairns & Pollock (1988).…”

“…Quantification of the fructan was determined by scanning the urea-spray-stained TLC foil (Gelscan, Pharmacia-LKB), on which standard concentrations of commercially available fructans (levan, Sigma) were also spotted. Sucrose, fructose and glucose levels were quantified by HPLC as described by Van der Meer et al (1994). Eor each transformed line at least 20 plants were used.…”