Cloning of the fructan biosynthesis pathway of Jerusalem artichoke

Abstract

To study the regulation of fructan synthesis in plants, we isolated two full-size cDNA clones encoding the two enzymes responsible for fructan biosynthesis in Jerusalem artichoke (Helianthus tuberosus): 1-sucrose:sucrose fructosyl transferase (1-SST) and 1-fructan:fructan fructosyl transferase (1-FFT). Both enzymes have recently been purified to homogeneity from Jerusalem artichoke tubers (Koops and Jonker (1994) J. Exp. Bot. 45, 1623-1631; Koops and Jonker (1996) Plant Physiol. 110, 1167-1175) and their amino acid sequences have been partially determined. Using RT-PCR and primers based on these sequences, specific fragments of the genes were amplified from tubers of Jerusalem artichoke. These fragments were used as probes to isolate the cDNAs encoding 1-SST and 1-FFT from a tuber-specific λZAP library. The deduced amino acid sequences of both cDNAs perfectly matched the sequences of the corresponding purified proteins. At the amino acid level, the cDNA sequences showed 61% homology to each other and 59% homology to tomato vacuolar invertase. Based on characteristics of the deduced amino acid sequence, the first 150 bp of both genes encode a putative vacuolar targeting signal. Southern blot hybridization revealed that both 1-SST and 1-FFT are likely to be encoded by single-copy genes. Expression studies based on RNA blot analysis showed organ-specific and developmental expression of both genes in growing tubers. Lower expression was detected in flowers and in stem. In other organs, including leaf, roots and dormant tubers, no expression could be detected. In tubers, the spatial and developmental expression correlates with the accumulation of fructans. Using the 1-sst and 1-fft cDNAs, chimeric genes were constructed driven by the CaMV 35S promoter. Analysis of transgenic petunia plants carrying these constructs showed that both cDNAs encode functional fructosyltransferase enzymes. Plants transformed with the 35S-1-sst construct accumulated the oligofructans 1-kestose (GF2), 1,1-nystose (GF3) and 1,1,1-fructosyl-nystose (GF4). Plants transformed with the 35S-1-fft construct did not accumulate fructans, probably because of the absence of suitable substrates for 1-FFT, i.e. fructans with a degree of polymerization ≤ 3 (GF2, GF3, etc.). Nevertheless, protein extracts from these transgenic plants were able to convert GF3, when added as a substrate, into fructans with a higher degree of polymerization. Progeny of crosses between a 35S-1-sst-containing 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 with those of other plant fructosyl transferase genes, we postulate that both plant fructan genes have evolved from plant invertase genes.