PURIFICATION, CLONING, AND FUNCTIONAL EXPRESSION OF SUCROSE-FRUCTAN 6-FRUCTOSYLTRANSFERASE, A KEY ENZYME OF FRUCTAN SYNTHESIS IN BARLEY

Fructans play an important role in assimilate partitioning and possibl y in stress tolerance in many plant families. Sucrose:fructan 6-fructo syltransferase (6-SFT), an enzyme catalyzing the formation and extensi on of beta-2,6-linked fructans typical of grasses, was purified from b arley (Hordeum vulgare L.). It occurred in two closely similar isoform s with indistinguishable catalytic properties, both consisting of two subunits with apparent masses of 49 and 23 kDa. Oligonucleotides, desi gned according to the sequences of tryptic peptides from the large sub unit, were used to amplify corresponding sequences from barley cDNA. T he main fragment generated was cloned and used to screen a barley cDNA expression library. The longest cDNA obtained was transiently express ed in Nicotiana plumbaginifolia protoplasts and shown to encode a func tional 6-SFT. The deduced amino acid sequence of the cDNA comprises bo th subunits of 6-SFT. it has high similarity to plant invertases and o ther beta-fructosyl hydrolases but only little to bacterial fructosylt ransferases catalyzing the same type of reaction as 6-SFT.