Synthesis of fructans by fructosyltransferase from the tuberous roots of Viguiera discolor (Asteraceae)

Sucrose:sucrose fructosyltransferase (SST) and fructan:fructan fructosyl-transferase (FFT) activities from crude extracts of tuberous roots of Viguiera discolor growing in a preserved area of cerrado were analyzed in 1995-1996. SST activity was characterized by the synthesis of 1-kestose from sucrose and FFT activity by the production of nystose from 1-kestose. The highest fructan-synthesizing activity was observed during early dormancy (autumn), when both (SST and FFT) activities were high. The increase in synthetic activity seemed to start during the fruiting phase in the summer, when SST activity was higher than in spring. During winter and at the beginning of sprouting, both activities declined. The in vitro synthesis of high molecular mass fructans from sucrose by enzymatic preparations from tuberous roots collected in summer showed that long incubations of up to 288 h produced consistently longer polymers which resembled those found in vivo with respect to chromatographic profiles.

inulin; fructosyltransferase; fructan biosynthesis; Viguiera discolor; Asteraceae

Authorship

N.M. Itaya

Secretaria do Meio Ambiente do Estado de São Paulo, Seção de Fisiologia e Bioquímica de Plantas, Instituto de BotânicaSecretaria do Meio Ambiente do Estado de São PauloSecretaria do Meio Ambiente do Estado de São Paulo, Seção de Fisiologia e Bioquímica de Plantas, Instituto de Botânica

R.C.L. Figueiredo-Ribeiro

M.S. Buckeridge

SCIMAGO INSTITUTIONS RANKINGS

Figures | Formulas

Figures (6)
Formulas (2)

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Figure 1
- Sucrose:sucrose fructosyltransferase (SST) (black columns) and fructan:fructan fructosyltransferase (FFT) (gray columns) activities of extracts prepared from the tuberous roots of Viguiera discolor harvested during different phenological phases in the cerrado of Moji-Guaçu (SP) from May 1995 to December 1996. LD = Late dormancy, ED = early dormancy, SP = sprouting, FR = fruiting.

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Figure 2
- Products obtained from sucrose (A), kestose (B) and nystose (C). The extract prepared from the tuberous roots of Viguiera discolor at the fruiting phase was incubated with 400 mM sucrose, 400 mM 1-kestose or 200 mM nystose at 30^oC. The products glucose, fructose, sucrose, 1-kestose, nystose and DP>4 were quantified. The consumption of the substrates used in each reaction mixture was not considered for the calculations, although they were detectable by HPLC.

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Figure 3
- TLC of fructo-oligosaccharides produced after 288 h incubation of an extract of the tuberous roots of Viguiera discolor with 400 mM sucrose (2), 400 mM 1-kestose (3) and 200 mM nystose (4). Fructo-oligosaccharides from tubers of Helianthus tuberosus (lanes 1 and 6) and an enzymatic extract from V. discolor without substrate (lane 5) were used as standards. Each lane contains approximately 50 µg fructose equivalents. DP = Degree of polymerization.

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Figure 4
- HPAEC/PAD analysis of fructans on a CarboPack PA-1 column produced after incubation of an enzyme preparation from the tuberous roots of Viguiera discolor collected at the fruiting phase with 400 mM sucrose. Incubation was performed for 288 h at 30^oC and products (in vitro) were compared with soluble neutral carbohydrates (SNC) extracted from the same tissue (in vivo). The enzyme preparation is equivalent to 325 µg protein/ml and SNC is equivalent to 1 mg fresh mass of tuberous roots. Control represents 0 min incubation. G = Glucose, F = fructose, S = sucrose, 1K = 1-kestose, N = nystose. Numbers indicate the estimated degree of polymerization.

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Figure 5
- HPAEC/PAD analysis of fructans on a CarboPack PA-100 column produced after incubation of an enzyme preparation from the tuberous roots of Viguiera discolor collected at the fruiting phase with 400 mM sucrose. Incubation was performed for 288 h at 30^oC and products (in vitro) were compared with fructans extracted from the same tissue (in vivo) and from tubers of H. tuberosus. Sample concentration was 4 mg/ml fructose equivalents. G = Glucose, F = fructose, S = sucrose, 1K = 1-kestose, N = nystose. Numbers indicate the estimated degree of polymerization.

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Figure 6
- Reaction scheme for reactions between sucrose and oligo- and polysaccharides within incubation media of the fructan synthesizing system of Viguiera discolor. Pentasaccharide is shown in a box because it is thought to be the principal substrate for FFT activity. SST = Sucrose:sucrose fructosyltransferase, FFT = fructan:fructan fructosyltransferase, FEH = fructan hydrolase.

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Figure 1 - Sucrose:sucrose fructosyltransferase (SST) (black columns) and fructan:fructan fructosyltransferase (FFT) (gray columns) activities of extracts prepared from the tuberous roots of Viguiera discolor harvested during different phenological phases in the cerrado of Moji-Guaçu (SP) from May 1995 to December 1996. LD = Late dormancy, ED = early dormancy, SP = sprouting, FR = fruiting.

Figure 2 - Products obtained from sucrose (A), kestose (B) and nystose (C). The extract prepared from the tuberous roots of Viguiera discolor at the fruiting phase was incubated with 400 mM sucrose, 400 mM 1-kestose or 200 mM nystose at 30^oC. The products glucose, fructose, sucrose, 1-kestose, nystose and DP>4 were quantified. The consumption of the substrates used in each reaction mixture was not considered for the calculations, although they were detectable by HPLC.

Figure 3 - TLC of fructo-oligosaccharides produced after 288 h incubation of an extract of the tuberous roots of Viguiera discolor with 400 mM sucrose (2), 400 mM 1-kestose (3) and 200 mM nystose (4). Fructo-oligosaccharides from tubers of Helianthus tuberosus (lanes 1 and 6) and an enzymatic extract from V. discolor without substrate (lane 5) were used as standards. Each lane contains approximately 50 µg fructose equivalents. DP = Degree of polymerization.

Figure 4 - HPAEC/PAD analysis of fructans on a CarboPack PA-1 column produced after incubation of an enzyme preparation from the tuberous roots of Viguiera discolor collected at the fruiting phase with 400 mM sucrose. Incubation was performed for 288 h at 30^oC and products (in vitro) were compared with soluble neutral carbohydrates (SNC) extracted from the same tissue (in vivo). The enzyme preparation is equivalent to 325 µg protein/ml and SNC is equivalent to 1 mg fresh mass of tuberous roots. Control represents 0 min incubation. G = Glucose, F = fructose, S = sucrose, 1K = 1-kestose, N = nystose. Numbers indicate the estimated degree of polymerization.

Figure 5 - HPAEC/PAD analysis of fructans on a CarboPack PA-100 column produced after incubation of an enzyme preparation from the tuberous roots of Viguiera discolor collected at the fruiting phase with 400 mM sucrose. Incubation was performed for 288 h at 30^oC and products (in vitro) were compared with fructans extracted from the same tissue (in vivo) and from tubers of H. tuberosus. Sample concentration was 4 mg/ml fructose equivalents. G = Glucose, F = fructose, S = sucrose, 1K = 1-kestose, N = nystose. Numbers indicate the estimated degree of polymerization.

Figure 6 - Reaction scheme for reactions between sucrose and oligo- and polysaccharides within incubation media of the fructan synthesizing system of Viguiera discolor. Pentasaccharide is shown in a box because it is thought to be the principal substrate for FFT activity. SST = Sucrose:sucrose fructosyltransferase, FFT = fructan:fructan fructosyltransferase, FEH = fructan hydrolase.

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Synthesis of fructans by fructosyltransferase from the tuberous roots of Viguiera discolor (Asteraceae)

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