Production of novel fructans through genetic engineering of crops and their applications

• Research unit: EU RFP
• Project number: 96.0139

Key words (English)

Fructan; sugar sensing; carbohydrate allocation; regulation; drought resistance; enzyme activity; structure-function

Alternative project number (English)

EU project number: FAIR-CT96-1896

Research programs (English)

EU-programme: 4. Frame Research Programme - 4.3 Biomedical/Health research

Short description (English)

See abstract

Partners and International Organizations (English)

University of Utrecht (NL), Instituto Nazionale per l'Analisi e Protezione degli Agroecosistemi, Firenze (I), Sensus, Roosendaal (NL), Advanta, Tienen (B)

Abstract (English)

We feel that the results obtained by our group are significant advances in the understanding of the structures and functions involved in fructan metabolism.
The main achievements of the Botanical Institute of the University of Basel during the 4½ year grant period according to objectives of Task 1 were the following:
1. Gene cloning and characterization: The functional expression of the barley 6-SFT in Pichia pastoris representing so-far the most suitable heterologous expression system for fructosyltransferases was a milestone for the project dealing with the structure-function analysis of these enzymes. This system furthermore offers the possibility to rapidly assay unknown sequences for activity. The successful purification of a 1-SST from barley leaf growth zones allowed after microsequencing of peptide fragments to help designing primers for a PCR-based cloning strategy. Subsequently, a 1-SST from Festuca arundinaceae (tall fescue) was cloned and functionally expressed in P.pastoris. This showed that in fescue most likely, separate genes, encoding the 1-SST and 6G-FFT activities that could not be separated using protein purification procedures earlier. We could show that addition of tags to the recombinant protein expressed in P.pastoris did not affect the activities of the barley 6-SFT neither qualitatively nor quantitatively. We then constructed a hybrid protein consisting of the N-terminal part of the mature fescue 1-SST and the tagged C-terminal part of the barley 6-SFT. The activity of this protein was tested in P. pastoris and corresponded exactly to the activity of a 1-SST. Therefore, the determinants for a specific activity reside on the N-terminal subunit of these proteins.
2. Regulation of fructan metabolism: To study the details of gene regulation, we isolated 1.6 kb of the genomic DNA sequence located 5' of the barley 6-SFT coding region. Using a promoter-reporter gene construct, (uidA encoding b-glucuronidase) and microprojectile bombardment, we were able to show that the cloned sequence contains the necessary cis-acting elements to confer light- and sucrose-mediated induction of 6-SFT transcription. The 6-SFT promoter contains elements described to confer light-dependent transcription. We performed experiments with the excised leaf system, using DCMU and EDTA to inhibit the accumulation of sugar in the tissue. We could show that 6-SFT mRNA and fructan did not accumulate upon illumination of the leaves but only if sucrose was supplied together with the inhibitors. Therefore sugar appears to be the main trigger of fructan synthesis already at the level of transcription.
Our contribution in the context of Task 4 (Biochemical evaluation) was limited to the analysis of primary tobacco 1-SST transformants. No fructans were found in these plants.

References in databases (English)

Swiss Database: Euro-DB of the
State Secretariat for Education and Research
Hallwylstrasse 4
CH-3003 Berne, Switzerland
Tel. [phone]
Swiss Project-Number: 96.0139