Isolation of galactinol from leaves of Cucumis sativus

Pharr, D. M.; Hendrix, Donald L.; Robbins, N. Suzanne; Gross, Ken; Sox, H. N.

doi:10.1016/0168-9452(87)90026-4

Cited by 11 publications

(12 citation statements)

References 6 publications

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“…The loliose synthetic pathway was determined by testing different galactosyl donors, galactinol (purified from cucumber [Cucumis sativus] according to Pharr et al [1987]), UDP-Gal, and raffinose (Sigma-Aldrich). The pH optimum of the enzyme reaction was determined using MES buffer (pH 5.5-6.7 adjusted with NaOH), HEPES buffer (pH 6.7-8 adjusted with NaOH), and HEPBS buffer (pH 8-9 adjusted with NaOH) with 50 mm Suc and 5 mm UDP-Gal as substrates.…”

Section: Loliose Synthase Characterizationmentioning

confidence: 99%

Fructans, But Not the Sucrosyl-Galactosides, Raffinose and Loliose, Are Affected by Drought Stress in Perennial Ryegrass

et al. 2003

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The aim of this study was to evaluate the putative role of the sucrosyl-galactosides, loliose [␣-d-Gal (1,3) ␣-d-Glc (1,2) ␤-d-Fru] and raffinose 6) ␣-d-Glc (1,2) ␤-d-Fru], in drought tolerance of perennial ryegrass and to compare it with that of fructans. To that end, the loliose biosynthetic pathway was first established and shown to operate by a UDP-Gal: sucrose (Suc) 3-galactosyltransferase, tentatively termed loliose synthase. Drought stress increased neither the concentrations of loliose and raffinose nor the activities of loliose synthase and raffinose synthase (EC 2.4.1.82). Moreover, the concentrations of the raffinose precursors, myoinositol and galactinol, as well as the gene expressions of myoinositol 1-phosphate synthase (EC 5.5.1.4) and galactinol synthase (EC 2.4.1.123) were either decreased or unaffected by drought stress. Taken together, these data are not in favor of an obvious role of sucrosyl-galactosides in drought tolerance of perennial ryegrass at the vegetative stage. By contrast, drought stress caused fructans to accumulate in leaf tissues, mainly in leaf sheaths and elongating leaf bases. This increase was mainly due to the accumulation of long-chain fructans (degree of polymerization Ͼ 8) and was not accompanied by a Suc increase. Interestingly, Suc but not fructan concentrations greatly increased in drought-stressed roots. Putative roles of fructans and sucrosyl-galactosides are discussed in relation to the acquisition of stress tolerance.One of the strategies employed by plants to survive drought stress includes the synthesis of protective compounds, which may act by stabilizing membranes and proteins or mediating osmotic adjustment (Bohnert et al., 1995; Hare et al., 1998; Hoekstra et al., 2001). Included among these protective compounds are the water-soluble carbohydrates (WSCs), Glc, Suc, raffinose, myoinositol, and fructans.Raffinose family oligosaccharides (RFOs) such as raffinose and stachyose accumulate during seed development and are thought to play a role in the desiccation tolerance of seeds (Blackman et al., 1992; Brenac et al., 1997). Raffinose also accumulates in vegetative tissues under drought stress (Taji et al., 2002). RFO biosynthesis requires the presence of galactinol, which is formed by galactinol synthase (GolS; EC 2.4.1.123) from UDP-Gal and myoinositol. Galactinol is the galactosyl donor for the biosynthesis of raffinose from Suc by raffinose synthase (RafS; EC 2.4.1.82). Because galactinol has not been assigned any function in plants other than acting as galactosyl donor for RFOs synthesis, GolS potentially catalyzes a metabolic key step for RFO synthesis. In a recent study, two drought-responsive GolS genes were identified among seven in Arabidopsis (Taji et al., 2002). Overexpression of one of them caused an increase in endogenous galactinol and raffinose as well as an improvement in drought tolerance.In addition to GolS, myoinositol 1-phosphate synthase (INPS; EC 5.5.1.4) is another enzyme that may control the levels of galactinol and raffinose. It repr...

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Section: Loliose Synthase Characterizationmentioning

confidence: 99%

Fructans, But Not the Sucrosyl-Galactosides, Raffinose and Loliose, Are Affected by Drought Stress in Perennial Ryegrass

et al. 2003

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“…LJDP-[U-'4C]Gal (1 1 .O GBq/mmol) was from New England Nuclear. Galactinol was isolated from A,iuga reptans or Cucurbita pepo Gelber Zentner leaves accortiing to the method of Pharr et al (1987), stachyose was purchased from Sigma, alnd verbascose was obtained from MegaZyme (North Rocks, New South Wales, Australia). Amyloglucosidase (from Aspergilhs niger), a-amylase (from Bacillus subtilis), and 0-D-Gal dehydrogenase (from Pseudomonas fluorescens) were purchased from Boehringer (Mannheim, Germany).…”

Section: Reagentsmentioning

confidence: 99%

Metabolism of the Raffinose Family Oligosaccharides in Leaves of Ajuga reptans L. (Cold Acclimation, Translocation, and Sink to Source Transition: Discovery of Chain Elongation Enzyme)

1994

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Aiuga reptans is a frost-hardy, perennial labiate that is known for its high content of raffinose family oligosaccharide(s) (RFO). Seasonal variations in soluble nonstructural carbohydrate levels in above-ground parts of Aiuga showed that the RFO were by far the most predominant components throughout the whole year. RFO were lowest in summer (75 mg/g fresh weight) and highest in fall/ winter (200 mg/g fresh weight), whereas sucrose and starch were only minor components. Cold treatment (14 d at 10/3"C, day/ night) of plants that were precultivated under warm conditions (25'C) lowered the temperature optimum of net photosynthesis from 16' to 8"C, decreased the maximum rate, and increased the total nonstructural carbohydrate content of leaves by a fador of about 10, mainly because of an increase of RFO. The degree of polymerization of the R F O increased sequentially up to at least 15. A novel, galactinol-independent galactosyltransferase enzyme was found, forming from two molecules of RFO, the next higher and lower degree of polymerization of RFO. The enzyme had a pH optimum of 4.5 to 5.0 and may be responsible for RFO chain elongation. RFO were the main carbohydrates translocated in the phloem, with stachyose being by far the most dominant form. Studies of carbon balance during leaf development revealed a transition point between import and export at approximately 25% maximal leaf area. RFO synthesis could be detected even before the commencement of export, suggesting the existence of a nonphloem-linked RFO pool even in very young leaves. Taken together, it seems that Ajuga leaves contain two pools of RFO metabolism, a pronounced long-term storage pool in the mesophyll, possibly also involved in frost resistance, and a transport pool in the phloem.RFO (a-1,6-galactosyln-Suc; 1 5 n < approximately 7) are the most widely occuning oligosaccharides in the plant kingdom. RFO synthesis proceeds by sequential action of a series of a-galactosyltransferases (Kandler and Hopf, 1984; Dey, been found (Hérissey et al., 1954) that are thought to be synthesized sequentially by specific galactosyltransferases using galactinol as the Gal donor analogously to the synthesis of raffinose and stachyose. RFO catabolism proceeds by sequential exo-action of a-Gal(s).Almost a11 biochemical and physiological studies conceming RFO metabolism have been performed on leaves of cucurbits and seeds of legumes. The specialization on these non-frost-hardy plants may lead to a bias with regard to the roles of the RFO in plant metabolism. The main functions of the RFO are still believed to be carbon transport in leaves and desiccation tolerance and carbon storage in seeds. Less attention has been given to equally important functions of the RFO such as cold tolerance (e.g. in conifer needles, Hinesley et al., 1992; Wiemken and Ineichen, 1993) and carbon storage (eg. in Stachys tubers, Keller and Matile, 1985) in vegetative plant parts. To increase our knowledge of RFO metabolism in these less well-studied areas, we needed a suitable experimental pl...

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“…The identity of galactinol was confirmed by the reaction of this compound with stachyose synthase (EC 2.4.1.67) to produce stachyose in the presence of raffinose The preparation of crude enzyme extracts and the analysis of stachyose formation followed the basic procedures described by Pharr et aI. (13). Briefly, 4 g of fresh, mature, dark green leaves harvested from zucchini plants, which were grown in a greenhouse for 5 wk as described previously (17), were homogenized with a mortar and pestle at 0°C in 10 mL of 50 mM of N-2-hydroxyethyl piperazine-N'-2 ethanesulfonic acid (HEPES)-NaOH, pH 7.0, containing 5 mM dithiothreitol (DTT).…”

Section: Methodsmentioning

confidence: 99%

“…1D). Table 1 summarizes the yield and purity of galactinol isolated from defatted castor meal and compares the procedure with that used to purify galactinol from mature cucumber leaves (13).…”

Section: Isolationmentioning

confidence: 99%

“…In contrast to the raffinose saccharides, galactinol is present at relatively low levels in various photosynthetic leaf tissues (4-6), soybean seeds (7-9), sugar beet molasses (10), and cold-stored potato tubers (11). While galactinol has been isolated from sugar beet molasses by Brown and Serro {10), and McCready et al (12), and from leaf extracts of Lamium maculatum by Senser and Kandler (6) and Cucumis sativus by Pharr et aL (13), it is unavailable commercially. FurtheP more, a routine method for obtaining sufficient quantities of galactinol is limited to a few laboratories where suitable source material, techniques and facilities are available Consequently, studies of the raffinose biosynthetic pathway, necessary for understanding plant carbon metabolism and improving food quality, have been hindered due to the unavailability of this metabolit~In a previous study (14), we have characterized soluble oligosaccharides in 33 different dry seeds by high-performance liquid chromatography (HPLC).…”

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Isolation and identification of galactinol from castor oilseed meal

Kuo

1992

J Americ Oil Chem Soc

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Galact~ol, 1-O~-I~galactopyr~l-~my~inositol, is an essential intermediate in the biosynthesis of raffinose sac ~ charides in plant tissues. The unavailability of a comme~ clal source for this metabolite has hindered research on raffinose saccharide metabolism to date. The objective of this study was to develop a facile method for obtaining highly purified galac¢inol from a readily available source. Defatted castor oilseed meal was found to exhibit a simple soluble carbohydrate profile that included galactinol. Purification of galactinol from castor meal was achieved by enzymatic treatment and a series of liquid chromatography steps, including ion-exchange and carbon adsorption. The isolation procedure was reliable with a yield of 278 mg of galactinol from 150 g of defatted meal. The purity of galac ~ tinol was estimated at 96.4% by high-performance liquid chromatography. The identity of the purified galactinol was confirmed by enzymatic analysis, identical relative retention times on high-performance liquid chromatography and gas chromatography, and comparison of its mass spectrum to that of authentic galactinol.KEY WORDS: Castor oilseed, chromatography, defatted meal, galactinol, invertase. (1), serves as the galactosyl donor in the formation d the abundant oligosaccharides of the raffinose family in higher plants (2,3). In contrast to the raffinose saccharides, galactinol is present at relatively low levels in various photosynthetic leaf tissues (4-6), soybean seeds (7-9), sugar beet molasses (10), and cold-stored potato tubers (11). While galactinol has been isolated from sugar beet molasses by Brown and Serro {10), and McCready et al. (12), and from leaf extracts of Lamium maculatum by Senser and Kandler (6) and Cucumis sativus by Pharr et aL (13), it is unavailable commercially. FurtheP more, a routine method for obtaining sufficient quantities of galactinol is limited to a few laboratories where suitable source material, techniques and facilities are available Consequently, studies of the raffinose biosynthetic pathway, necessary for understanding plant carbon metabolism and improving food quality, have been hindered due to the unavailability of this metabolit~ In a previous study (14), we have characterized soluble oligosaccharides in 33 different dry seeds by high-performance liquid chromatography (HPLC). An unknown peak with a retention time similar to authentic galactinol was present in several different seed extracts. The objectives of the present study were to confirm the identity of galactinol in one of these seed extracts, namely castor bean (Ricinus communis), and to develop an effective method for its purification from castor meal. Galactinol, 1-O-a-D-galactopyranosyl-D-myo-inositol

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Isolation of galactinol from leaves of Cucumis sativus

Cited by 11 publications

References 6 publications

Fructans, But Not the Sucrosyl-Galactosides, Raffinose and Loliose, Are Affected by Drought Stress in Perennial Ryegrass

Fructans, But Not the Sucrosyl-Galactosides, Raffinose and Loliose, Are Affected by Drought Stress in Perennial Ryegrass

Metabolism of the Raffinose Family Oligosaccharides in Leaves of Ajuga reptans L. (Cold Acclimation, Translocation, and Sink to Source Transition: Discovery of Chain Elongation Enzyme)

Isolation and identification of galactinol from castor oilseed meal

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