CLONING OF THE FRUCTAN BIOSYNTHESIS PATHWAY OF JERUSALEM-ARTICHOKE

To study the regulation of fructan synthesis in plants, we isolated tw o full-size cDNA clones encoding the two enzymes responsible for fruct an biosynthesis in Jerusalem artichoke (Helianthus tuberosus): 1-sucro se:sucrose fructosyl transferase (1-FFT) and 1-fructan:fructan fructos yl transferase (1-FFT). Both enzymes have recently been purified to ho mogeneity from Jerusalem artichoke tubers (Koops and Jonker (1994) J. Exp. Bot. 45, 1623-1631; Koops and Jonker (1996) Plant Physiol. 110, 1 167-1175) and their amino acid sequences have been partially determine d. Using RT-PCR and primers based on these sequences, specific fragmen ts of the genes were amplified from tubers of Jerusalem artichoke. The se fragments were used as probes to isolate the cDNAs encoding 1-SST a nd 1-FFT from a tuber-specific lambda ZAP library. The deduced amino a cid sequences of both cDNAs perfectly matched the sequences of the cor responding purified proteins. At the amino acid level, the cDNA sequen ces showed 61% homology to each other and 59% homology to tomato vacuo lar invertase. Based on characteristics of the deduced amino acid sequ ence, the first 150 bp of both genes encode a putative vacuolar target ing signal. Southern blot hybridization revealed that both 1-SST and I -FFT are likely to be encoded by single-copy genes. Expression studies based on RNA blot analysis showed organ-specific and developmental ex pression of both genes in growing tubers. Lower expression was detecte d in flowers and in stem. In other organs, including leaf, roots and d ormant tubers, no expression could be detected. In tubers, the spatial and developmental expression correlates with the accumulation of fruc tans. Using the 1-sst and 1-fft cDNAs, chimeric genes were constructed driven by the CaMV 35S promoter. Analysis of transgenic petunia plant s carrying these constructs showed that both cDNAs encode functional f ructosyltransferase enzymes. Plants transformed with the 35S-1-sst con struct accumulated the oligofructans 1-kestose (GF(2)), 1,1-nystose (G FB) and 1,1,1-fructosyl-nystose (GF(4)). Plants transformed with the 3 5S-1-fft construct did not accumulate fructans, probably because of th e absence of suitable substrates for 1-FFT, i.e. fructans with a degre e of polymerization greater than or equal to 3 (GF(2), GF(3), etc.). N evertheless, protein extracts from these transgenic plants were able t o convert GF(3), when added as a substrate, into fructans with a highe r degree of polymerization. Progeny of crosses between a 35S-1-sst-con taining plant and a 35S-1-fft-containing plant, showed accumulation of high-molecular-weight fructans in old, senescent leaves. Based on the comparison of the predicted amino acid sequences of 1-sst and 1-fft w ith those of other plant fructosyl transferase genes, we postulate tha t both plant fructan genes have evolved from plant invertase genes.