The content, distribution and structure of fructo- oligosaccharides in onion plants were investigated extensively by the researchers cited above, although.
New Phytol. (1997), 136, 105-113
Fructo-oligosaccharide content and fructosyltransferase activity during growth of onion bulbs BY N O R I O S H I O M I * , S H U I C H I
ONODERA
AND H I D E K I
Department of Food Science, Faculty of Dairy Science, Rakuno Gakuen 582 Bunkyodai, Midorimachi, Ebetsu, Hokkaido 069, Japan
SAKAI
University,
{Received 24 July 1996)
SUMMARY Tbe accumulation of fructo-oligosaccbarides and tbe activities of fructosyltransferase (sucrose: sucrose lpfructosyltransferase (SST), l^-fructosyltransferase (lp-FT) and 6fj-fructosyltransferase (6g-FT)) in tbe bulbs of tbree onion cultivars were investigated from June to September 1993. Tbe total fructo-oligosaccbaride content increased from June until August, tben decreased in September, except in one cultivar. Tbe levels of neokestose and its related tetrasaccbarides (lp,6Q-di-/5-D-fructofuranosyl sucrose and 6(j(l-y5-D-fructofuranosyl)2Sucrose) were bigber tban tbose of 1-kestose and nystose tbrougbout growtb. Tbe activities of 6(j-FT, l^-FT and SST were bigb in June and July, tben decreased; SST activity was very low in September. Tbe activity ratios of 6g-FT to lj,F T varied between 1-86 and 2-65 over tbe growtb period. Two trisaccbarides, tbree tetrasaccbarides and four pentasaccbarides were identified, togetber witb a mixture of bexa- and beptasaccbarides, all of wbicb were syntbesized in vitro from 0-1 M sucrose by an enzyme preparation of onion bulbs barvested in August. Octa- and nonasaccbarides otber tban tbe saccbarides formed from sucrose were also syntbesized from O'l M 1-kestose or 0-1 M neokestose. All tbe saccbarides produced from sucrose, 1-kestose or neokestose by tbe crude enzyme prepared from onion bulbs were identical to tbe saccbarides occurring naturally in onion bulbs. Key words: Allium cepa, fructo-oligosaccbarides, fructosyltransferase.
comprise 1-kestose and neokestose (Bacon, 1959; Darbyshire & Henry, 1978). The tetra-, penta-, and hexasaccharide fractions Fructose-oligomers and fructose-polymers are major reserve carbohydrates in higher plants such as from onion leaves and bulbs were reported recently members of the Asteraceae, Liliaceae and to comprise nystose, 6(j(l-/?-fructofuranosyl)2SucGramineae. Fructose-ohgomers (fructo-oligo- rose and lp,6(j-di-^-fructofuranosylsucrose; Ip(l-y5saccharides (DP 3-c. 10)) together with glucose, fructofuranosyOgSucrose, 6Q(1 -/?-fructofuranosyl)3fructose and sucrose are contained in onion, which is l p-/?-fructofuranosyl-6(j(l-/^-fructofuranoone of the most widely distributed liliaceous plants in the world (Bacon, 1957, 1959; Bose & Shrivastava, tofuranosyl sucrose; and lp(l-y^-fructofuranosyl)n1961; Darbyshire & Henry, 1978). The distribution 5^(l_^_fructofuranosyl)j^ sucrose (n + m = 4), reof fructo-oligosaccharides in onion leaf-bases has spectively (Shiomi, 1989; Shiomi et al, 1991). The content, distribution and structure of fructobeen studied, and fructo-oligosaccharide contents were found to be higher in younger leaf-bases (inner) oligosaccharides in onion plants were investigated than in older (outer) leaf-bases (Bacon, 1959; extensively by the researchers cited above, although older studies were carried out using techniques such Darbyshire & Henry, 1978). The composition of fructo-oligosaccharide iso- as paper chromatography (PC), thin-layer chromatomers in onion plants was also investigated, and the graphy (TLC), and high-performance liquid trisaccharide fraction from onion bulbs was shown to chromatography (HPLC) with refractive index monitoring. However, little is known about the changes in fructo-oligosaccharide isomers derived from 1-kestose and neokestose, and in the activities of fructosyltransferases (sucrose: sucrose lpINTRODUCTION
'• To whom correspondence should be addressed.
106
N. Shiomi, S. Onodera and H. Sakai
fructosyltransferase (SST, EC 2.4.1.99; Henry & Darbyshire, 1980), 6c.-fructosyltransferase (6ct-FT; Shiomi, 1989) and 1 p-fructosyltransferase (lj,-FT; Henry & Darbyshire, 1980; Shiomi, 1989)) involved in the synthesis of fructo-oligosaccharides in onion bulbs during the growth stage. Here, we describe the accumulation of fructooligosaccharide isomers and the activities of fructosyltransferases (SST, 6 Q - F T and l^-FT) in bulbs of three onion cultivars during the growth period, and study the synthesis in vitro of fructooligosaccharide isomers from sucrose, 1-kestose or neokestose by a crude enzyme preparation from onion bulbs.
MATERIALS AND METHODS
mixtures were separated on an HPLC-carbohydrate column (PAl, Carbo Pack, Sunnyvale, CA, USA) with a Dionex Bio LC series HPLC using pulsed amperometric detection (PAD) (Rocklin & Pohl, 1983; Johnson, 1986). The gradient was established by mixing eluent A (150 mM NaOH) with eluent B (500 mM sodium acetate in 150 mM NaOH) in the following two ways: System 1: 0-1 min, 25 mM; 1-2 min, 25-50 mM; 2-20 min, 50-200 mM; 20-22 min, 500 mM; 22-30 min, 25 mM. System H: 0-1 min, 5mM; 1-2 min, 25-50 mM; 2-14 min, 50-500 mM; 14-22 min, 500 mM; 22-30 min, 25 mM; flow rate through the column was 1-0 ml min"^ The applied PAD potentials for El (500 ms), E2 (100 ms) and E3 (50 ms) were 0-01, 0-60 and -0-6 V, respectively, and the output range was 1 juC.
Paper and thin-layer chromatography (PC and TLC). Two solvents, «-propanol-ethyl acetate-water Materials (7:1:2) (I) and w-butanol-acetic acid-water (4:1 :2) Bulbs of onion {Allium cepa L. cv. Sapporo Yellow (H), were used for paper chromatography (filter (SY); cv. Southport White Glove Highsolid paper No. 50, Toyo, Tokyo, Japan), and a third (SWGH) and cv. Sweet Spanish Utah Jumbo solvent, w-butanol-isopropanol-water (10:5:4) (HI), (SSUT)) were harvested at the experimental farm of was employed for thin-layer chromatography the Hokkaido National Agricultural Experiment (Kieselgel 60F254 pre-coated, Merck, Darmstadt, Station on 25 June, 28 July, 26 August and 27 Germany). After three to five developments, the September 1993, and stored frozen. Standard sugars chromatograms were sprayed with anisidine phoswere prepared as follows. Crystalline 1-kestose (3a: phate solution for detecting sugars (Mukherjee & lp-/?-D-fructofuranosylsucrose) and nystose (4 a: Srivastava, 1952). lp(l-y^-D-fructofuranosyl)2Sucrose) were prepared from sucrose by a Scopulariopsis brevicaulis enzyme (Takeda et al., 1994). 6-Kestose was synthesized via Preparation of enzyme solution the transfer action of yeast invertase (grade V H , Sigma Chemicals, St Louis, M O , USA) on sucrose. All the operations for enzyme preparation were Neokestose (3 b : 6c,-/?-D-fructofuranosylsucrose) carried out at 4 °C, and centrifugation was conducted and lp(l-/?-D-fructofuranosyl)^-6Q (l-/?-D-fructo- at 10000^ for 30 min. Onion bulbs (five globes, or furanosyl)^ sucrose ( 4 b : w? = 0, w = 2; 4 c : m = l . 20 globes harvested in June) were peeled, cut into small pieces and mixed thorough^. The pieces (50 g) 2, n=\; 5 d : m = l , n = 2\ 6 b : m = 0, w = 4 ; 6 c : were homogenized in 0-05 M phosphate buffer, pH 7-0 (150 ml). The homogenate was filtered through 8:n + m = 6,9x:w + m ^ 7) were obtained from aspa- cheesecloth. The residue was homogenized twice ragus roots as described in previous papers (Shiomi, more, and the resulting filtrate was centrifuged. The Yamada & Izawa, 1976, 1979; Shiomi, 1981). 1^(1- supernatant was made up to 500 ml with the same /?-D-Fructofuranosyl)^sucrose(m — 4(6 a) and 5(7 a)) buffer, 50 ml was centrifuged, dialysed for 1 d against stirred 0-01 M phosphate buffer, pH 6-1, 5 1 were prepared from Jerusalem artichoke tubers. and was concentrated to 2 ml using a Centriprep 30 (Amicon Beverly, MA, USA). The concentrate was diluted to 50 ml with 0-01 M phosphate buffer then Quantitative determination of sugars re-concentrated as above, and the process repeated. Total sugars were determined by the anthrone The concentrate was termed 'supernatant concenmethod (Morris, 1948) and reducing sugars were trate A' and was assayed for enzyme activities (SST, measured by the method of Somogyi and Nelson lp-FT, 6(j-FT and invertase). For studying the synthesis of fructo(Nelson, 1944; Somogyi, 1945). oligosaccharides from sucrose, 1-kestose or neokestose, a supernatant concentrate was prepared independently from onion bulbs (' Sapporo Yellow') Analysis of sugars harvested on 26 August 1993. The flve bulbs were High-performance anion-exchange chromatography peeled and cut into small pieces which were mixed (HPAEC). The synthesized or extracted saccharide thoroughly. The supernatant (900 ml) obtained from
Fructans in onion bulbs the onion pieces (100 g) was dialysed, concentrated to 2-5 ml by ultrafiltration apparatus (PM-10 membrane, Amicon) and Centriprep30, and termed 'supernatant concentrate B'. Supernatant concentrates A and B were prepared in duplicate and assayed for enzyme activity. Measurement of enzyme activity SST activity. One kat of SST activity was defined as the amount of enzyme which catalysed fructosyl transfer from sucrose to sucrose to produce 1 mole of 1-kestose (isokestose, 3a) in 1 s under the conditions described below. A mixture (I) of enzyme (04 ml), 0-4 M sucrose (0-2 ml), Mcllvaine buffer (pH 5-5, 0-2 ml) and toluene (a trace amount) was incubated at 30 °C for 5, 10 and 15 h. After the reaction was stopped by heating in a boiling water bath for 3-5 min, the reaction mixture was diluted, filtered and applied to HPAEC. SST activity was calculated from the amount of 1-kestose. lp-FT activity. One kat of Ip-FT activity was defined as the amount of enzyme which catalysed the fructosyl transfer from 1 -kestose to another 1 -kestose to synthesis 1 mol of nystose (4 a) in 1 s under the conditions described below. The enzyme reactions were carried out at 30 °C for 5 and 10 min. A mixture (II) of enz3^me (0-4 ml), 04 M 1-kestose (0-2 ml) and Mcllvaine buffer (pH 5'5, 0-2 ml) was used. The reaction was terminated by heating in a boiling water bath for 3'5 min, and the reaction mixture was diluted, filtered and appHed to HPAEC. l^-ET activities were calculated from the amount of nystose (4a).
107 70 % ethanol (80 ml) containing small amounts of calcium carbonate, and were then immediately boiled in a water bath for 10 min. The residues were extracted five times in the same manner, then extracted with hot water (80 °C, 100 ml). Filtered extracts (25 ml) were concentrated in vacuo at 30-33 °C to dryness. The concentrated sugar was added to distilled water (25 ml) and the solution was deionized with Amberlite IRA-410. This process was duplicated.
RESULTS
Contents of saccharides in onion bulbs during the growth period The neutral sugar extracts from onion bulbs of all three cultivars harvested in September were analysed by HPAEC. As shown in Figure 1, peaks corresponding to glucose, fructose, sucrose and fructooligosaccharides isomers (3a, 1-kestose; 3b, neokestose and lp(l-y5-D-fructofuranosyl)^-6Q(l-/5'-Dfructofuranosyl)^sucrose (4a: m = 2, « = 0; 4b: m = 0, n = 2; 4 c : m = 1, « = 1 ; 5 a : m = 3, n — 0; 5h: m = 0, n = 3; 5c: m = 2, n = 1; 5d: m = 1, n =
2; 6a: m = 4, n = 0; 6b: m = 0, w = 4; 6 c : m = 3, n = 1; 6 d j : m = \, n = 3 ; d6.2'. m — 2, n = 2-,1 a: m = 5, n = 0; S: n + m = 6,9x: n + m^7)) w^ere detected on the chromatogram of SY onion bulbs. The chromatograms of sugar extracts from three other cultivars were qualitatively similar to that of the extract from SY onion bulbs. The decrease in the abundance of each successive oligosaccharide was
60-FT activity. One kat of 6(j-FT activity was defined as the amount of enzyme which catalysed the fructosyl transfer from 1 -kestose to another 1 -kestose to yield 1 mol of lp,6(j-di-/5-D-fructofuranosylsucrose (4c) in 1 s. The activities of 6 Q - E T were tested using mixture (II). All the experiments were made similarly to those for Ip-ET activity, except for calculating 6 Q - F T activity from the amount of 1J-,6Qdi-yS'-D-fructofuranosylsucrose (4 c). Invertase activity. One kat of invertase activity was defined as the amount of enzyme which hydrolyses 1 mol sucrose s~^. The experimental conditions were those described for SST measurements. Invertase activity was calculated from the amount of fructose released. Extraction of sugars Onion bulbs (five globes, or 20 globes if harvested in June) harvested between June and September were peeled and cut into small pieces which were mixed thoroughly. The pieces (10 g) were homogenized in
Retention time (min)
Figure 1. High-performance anion-exchange chromatogram of neutral soluble carbohydrate extracted from onion bulbs in September. G, glucose; F, fructose; S, sucrose; 3a, 1-kestose; 3b, neokestose; 4a, nystose; ly(l/?-D-fructofuranosyl)„,-6Q(l-/?-D-fructofuranosyl)„sucrose (4b: m = 0, n = 2; 4 c : m=\, n=\\ 5 a : m = 3, n = 0; 5b: m = 0, n = 3; 5 c : m = 2,n=\; 5 d : m = \,n = 2; 6 a : m = A, n = 0; 6 b : m = 0, n = 4; 6 c : m = 3, n = \; bd^. m= l,n = 3; 6d2: m = 2, n = 2; 7: m + n = 5; S: m->rn =
6; 9x: n + m'^1). The saccharides were eluted with system I (see text).
108
N. Shiomi, S. Onodera and H. Sakai 10
3-5
1-0
SY(a) 3-0
- 8
0-8 -
- 6
0-6 -
4
0-4 -
2-5 -
2-0 -
m
J/
~~'-'i3 3b"
^
5~H
1-5 -
4f
y4c~
;;.c:-'=X4b
1-0 -
/
' Jr'" ^ "^ ^ •" "
„ » — ^*
~'0
3a
02 -
- 2 0-5 -
0-0
4-0 -
1
1
1
1
1
I
1
1
0
0-0
10
1-0
5~H
1
I
I
o o
\
0-8
o OJ
3-0 6
o o ^
o §
0-6
Q.
a
CD
a 2.0
I
#
CD
- 4
2
CO
o
sz o ro o
1
SWGH {b)
SWGH (a) - 8
^
r
1
0-4
TJ >
.-
