Cloning and functional analysis of sucrose:sucrose 1-fructosyltransferase from tall fescue

Abstract

Enzymes of grasses involved in fructan synthesis are of interest since they play a major role in assimilate partitioning and allocation, for instance in the leaf growth zone. Several fructosyltransferases from tall fescue (Festuca arundinacea) have previously been purified (Lüscher and Nelson, 1995). It is surprising that all of these enzyme preparations appeared to act both as sucrose (Suc):Suc 1-fructosyl transferases (1-SST) and as fructan:fructan 6(G)-fructosyl transferases. Here we report the cloning of a cDNA corresponding to the predominant protein in one of the fructosyl transferase preparations, its transient expression in tobacco protoplasts, and its functional analysis in the methylotrophic yeast, Pichia pastoris. When the cDNA was transiently expressed in tobacco protoplasts, the corresponding enzyme preparations produced 1-kestose from Suc, showing that the cDNA encodes a 1-SST. When the cDNA was expressed in P. pastoris, the recombinant protein had all the properties of known 1-SSTs, namely 1-kestose production, moderate nystose production, lack of 6-kestose production, and fructan exohydrolase activity with 1-kestose as the substrate. The physical properties were similar to those of the previously purified enzyme, except for its apparent lack of fructan:fructan 6(G)-fructosyl transferase activity. The expression pattern of the corresponding mRNA was studied in different zones of the growing leaves, and it was shown that transcript levels matched the 1-SST activity and fructan content.

Figure 1

Schematic representation of the 1-SST cloning from tall fescue. Partial cDNA sequences of the tall fescue fructosyltransferase were obtained in five steps: two RT PCRs, 5′- and 3′-RACE, and PCR with genomic mini-plasmid libraries. DNA sequences are in black lines and gray lines represent the missing nucleotide sequence. All attempts to obtain the full-length cDNA with reverse PCR failed. Three short PCR fragments were cloned, cut out, and then re-assembled to a complete cDNA. The positions of the restriction sites KpnI and AvaI are marked. Arrows represent the positions of primers that were used for cloning or to obtain partial cDNAs. Vertical arrows show the positions of the two introns. The translational start and stop codons and the N terminus are marked.

Figure 2

Comparison of the deduced amino acid sequence of 1-SST from tall fescue (EMBL accession no. AJ297369) with the 6-SFT from barley (hv6sft: EMBL accession no. X83233), the 1-SST from Jerusalem artichoke (ht1sst: EMBL accession no. AJ009757), Allium cepa (ac1sst: EMBL accession no. AJ006066), and chicory (ci1sst: EMBL accession no. U81520), the 1-FFT from Jerusalem artichoke (htfft: EMBL accession no. AJ009756), and with the 6G-FFT from A. cepa (ac6gfft: EMBL accession no. Y07838). Identical amino acids are highlighted with a black background and similar amino acids with a gray background. The N terminus of the 58-kD protein is marked with an asterisk.

Figure 3

HPLC analysis of products formed in enzyme extracts from tobacco protoplasts transiently expressing the 1-SST clone from tall fescue (A and B), or the control with the empty plasmid (C), for 0 h (A), and 12 h (B and C), after incubation with 100 mm Suc for 1 h. G, Glc; F, Fru; S, Suc; I, 1-kestose.

Figure 4

Ratio of 1-SST and invertase activities in extracts from tobacco protoplasts transiently expressing the 1-SST clone from tall fescue for different time periods. The extracts were incubated with 100 mm Suc for 1 h. The ratio of 1-SST and invertase activity in the extracts is expressed as the ratio of 1-kestose and Fru production.

Figure 5

Transcript level of 1-SST in the base of the growing leaf, estimated by RT-PCR. Eight 1-cm-long segments were cut, starting from the base of each growing leaf, and the total RNA was isolated from each segment. A DNA fragment of 520 bp was amplified by RT-PCR from each sample (lanes 1–8) reflecting relative amounts of the 1-SST message (A), and analyzed by gel electrophoresis. As a control a 258-bp fragment of the rRNA was amplified from each sample (B).

Figure 6

SDS-PAGE analysis of concentrated culture medium from transformed P. pastoris expressing tall fescue 1-SST. Concentrated culture medium containing the recombinant 1-SST was separated on a 12% (w/v) gel by SDS-PAGE, and stained with Coomassie Brilliant Blue (lane FT). The control lane P0 contained concentrated medium from P. pastoris transformed with empty plasmid. M_r markers are shown on the right. The arrow at the left indicates the prominent single band of 82 kD.

Figure 7

Characterization of the recombinant tall fescue 1-SST produced by P. pastoris. A, Time course over a period of 0 to 210 min. 1-SST was incubated with 100 mm Suc as the substrate, and production of Fru, 1-kestose, and nystose was measured. B, Saturation experiment. Recombinant 1-SST was incubated for 60 min with Suc as the substrate with concentrations ranging from 0 to 1,000 mm. Fru, 1-kestose, and nystose production was measured. C, Influence of temperature on enzyme specificity. Enzyme preparation was incubated with 100 mm Suc under the following conditions: 0°C for 60 min, 0°C for 240 min, and 27°C for 60 min. Production of Fru and 1-kestose was determined.