Fructan Biosynthesis Research Articles

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

Purification and characterization of 1‐SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1‐SST (sucrose:sucrose 1‐fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots (Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63‐fold purification and a specific activity of 4.75 U (mg protein)−1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS‐PAGE apparent molecular masses of 49 kDa (α‐subunit) and 24 kDa (β‐subunit) were found. Further specification was obtained by MALDI‐TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS‐boiled chicory root extract and chicken‐raised polyclonal antibodies against the purified 1‐SST, indicating that the enzyme is a heterodimer in vivo. The N‐terminus of chicory root 1‐SST α‐subunit was shown to be highly homologous with the cDNA‐derived amino acid sequences from barley 6‐SFT and a number of β‐fructosyl hydrolases (in‐vertases and fructan hydrolases). However, chicory root 1‐SST properties could be clearly differentiated from those of chicory root 1‐FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1‐kes‐tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1‐SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1‐FFT and barley 6‐SFT, the enzyme also showed some β‐fructofuranosi‐dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β‐fructofuranosidase activity were also detected using 1‐kestose as the sole substrate.

Purification and Characterization of the Enzymes of Fructan Biosynthesis in Tubers of Helianthus tuberosus Colombia (II. Purification of Sucrose:Sucrose 1-Fructosyltransferase and Reconstitution of Fructan Synthesis in Vitro with Purified Sucrose:Sucrose 1-Fructosyltransferase and Fructan:Fructan 1-Fructosyltransferase).

Sucrose:sucrose 1-fructosyltransferase (1-SST), an enzyme involved in fructan biosynthesis, was purified to homogeneity from tubers of Helianthus tuberosus that were harvested in the accumulation phase. Gel filtration under native conditions predicted a molecular mass of about 67 kD. Electrophoresis or gel filtration under denaturing conditions yielded a 27- and a 55-kD fragment. 1-SST preferentially catalyzed the conversion of sucrose into the trisaccharide 1-kestose (GF2). Other reactions catalyzed by 1-SST at a lower rate were self-transfructosylations with GF2 and 1,1-nystose (GF3) as substrates yielding GF3 and 1,1,1-fructosylnystose, respectively, as products. 1-SST also catalyzed the removal of the terminal fructosyl unit from both GF2 and GF3, which resulted in the release of sucrose and GF2, respectively, and free Fru. The purified enzyme did not display [beta]-fructosidase activity. An enzyme mixture of purified 1-SST and fructan:fructan 1-fructosyltransferase, both isolated from tubers, was able to synthesize fructans up to a degree of polymerization of at least 13 with sucrose as a sole substrate.

Fructan biosynthesis in excised leaves of wheat (Triticum aestivum L.): a comparison of de novo synthesis in vivo and in vitro.

Carbohydrate accumulation by excised, continuously illuminated leaves of wheat (Triticum aestivum L.) was followed over a 24 h period. At 0 h, the tissue contained no detectable fructan. In the initial 6 h, only sucrose was accumulated. After 6 h the de novo synthesis of fructans was induced. Fructans accumulated in the sequence 1-kestose, bifurcose, nystose, oligofructans of apparent degree of polymerization (DP) up to 9 and finally, 6-kestose, which was first detected after 22 h. A cell-free protein extract from leaves illuminated for 24 h catalyzed the de novo synthesis of fructan from sucrose. The properties of this fructan synthetic activity (FSA) were characterized. The FSA was stable, exhibiting < 20% loss of activity when stored at 5 °C or 25 °C for 6 h. The FSA exhibited an apparent Km,suc of 114 mM, and an apparent pH optimum at 5.5. The in vitro synthesis of fructan of DP > 3 was not inhibited by sucrose even at 1000 mM. Pyridoxal-hydrochloride at 20 mM did not enhance rates of enzymatic fructan synthesis or significantly inhibit the release of free fructose in the optimized enzymatic reaction. The rate of oligofructan synthesis in the optimized reaction approximated to rates of accumulation in the leaf (1.35 mg g h-1 and 1.18 mg g h-1 respectively). The sequence of oligofructan synthesis in vitro was the same as that observed in the leaf, with the exception that 6-kestose was synthesized early in the time course, in parallel with 1-kestose and bifurcose. Fructans of apparent DP ≤ 8 were detected after 10 h of incubation. When incubated with bifurcose as sole substrate, the cell-free preparation liberated free monosaccharides, without the accumulation of trisaccharide or sucrose as intermediates. The results are discussed with reference to current, conflicting models for the biosynthesis of fructan in cereals.

Fructan biosynthesis in excised leaves of Lolium temulentum L.

SUMMARYA crude enzyme preparation from excised illuminated leaves of Lolium temulentum L. catalyzed the de novo synthesis of fructan of apparent degree of polymerization 3–20.In the absence of sucrose, the fructan synthetic activity (FSA) was labile at temperatures above 10 °C, but could be stored at 5 °C for 6 h with the loss of only 20% of the initial activity. Sucrose stabilized the FSA such that high rates of fructan synthesis continued for at least 6 h at 30 °C. By comparison, in the absence of sucrose 80% of the FSA was lost in 2 h at 30 °C.The FSA was maximal at pH 6·0 in citrate‐phosphate buffer and at 43 °C. It was not possible to saturate the FSA even at 1500 mol m−3 sucrose, or to calculate meaningful kinetic parameters for enzymatic fructan synthesis. 150 mill m−3 sucrose was the highest substrate concentration practically feasible and imposed an arbitrary maximum concentration for use in enzyme assays. Pyridoxal‐hydrochloride was found to reduce invertase activity by 40%, but also inhibited FSA by 20% at 1500 mol −3 sucrose and pH 6.0.

Structure of fructan oligomers in cheatgrass (Bromus tectorum L.) *

SUMMARYLeaves of field‐grown cheatgrass (Bromus tectorum L.) plants were sampled during early spring when day‐ and night‐time temperatures were relatively cool. Fructan oligomers with a degree of polymerization (DP) 3–6 were extracted and purified using gel and anion‐exchange chromatography. The structures of 13 cheatgrass fructans were established. They included two trisaccharides [1‐kestotriose (1‐kestose) and 6‐kestotriose (6‐kestose)], four tetrasaccharides [(1.1)‐kestotetraose (nystose), (1&amp;6)‐kestotetraose (bifurcose), (6,1)‐kestotetraose and (6,6)‐kestotetraose], three pentasaccharides [(1,1&amp;6)‐kestopentaose, (1&amp;6,6)‐kestopentaose and (6;1&amp;6)‐kestopentaose] plus four hexasaccharides [(1,1,1&amp;6)‐kestohexaose (1&amp;6,6,6)‐kestohexaose, (6;1&amp;6,6)‐kestohexaose and (6;1,6&amp;6)‐kestohexaose]. All fructans larger then DP 4 contained a branch point. Each member of the dominant series(1&amp;6)‐kestotetraose, (1&amp;6,6)‐kestopentaose and (1&amp;6,6,6)‐kestohexaose, is a branched fructan in which two fructose moieties are linked to the fructose subunit of each sucrose molecule. Thus the dominant series is built upon (1&amp;6)‐kestotetraose. Fructans larger than DP 3 with exclusively 2→1‐ or 2→6‐linkages were absent except for a very small amount of (6,6)‐kestotetraose. The unique fructan structures synthesized in cheatgrass, wheat and oats illustrate diversity in the enzymology of fructan biosynthesis among grass species.

Purification and characterization of three soluble invertases from barley (Hordeum vulgare L.) leaves.

Three soluble isoforms of invertase (beta-fructofuranosidase; EC 3.2.1.26) were purified from 7-d-old primary leaves of barley (Hordeum vulgare L.). Invertase I, a monomeric protein of 64 kD, was purified to apparent homogeneity as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Invertases IIA and IIB, multimeric proteins with molecular masses of the 116 and 155 kD, were purified 780- and 1370-fold, respectively, but were not yet homogeneous. Extracts of epidermal strips of leaves contained only invertase IIB. The specific activity of invertase was more than 100-fold higher in the epidermis than in the mesophyll. All three isoforms were acidic invertases, with pH optima of around 5.0 and little activity in the alkaline range. Invertase I had a Km for sucrose of 8.1 mM, and invertases IIA and IIB had much lower values of 1.0 and 1.7 mM, respectively. Invertase I was more than 2-fold more resistant than the other two invertases to the inhibitors HgCl2 and pyridoxal. All three constitutive invertases were found to act also as sucrose-sucrose fructosyltransferases when supplied with high concentrations of sucrose, forming 1-kestose as principal product. However, the fructosyltransferase activity of all three enzymes was inhibited by pyridoxal in the same way as their invertase activity. This characteristic clearly differentiates them from the inducible sucrose-sucrose fructosyltransferase of barley leaves, the activity responsible for the initial steps of fructan biosynthesis, which has previously been shown to be insensitive to pyridoxal.

Purification and properties of an invertase with sucrose: sucrose fructosyltransferase (SST) activity from the roots of Cichorium intybus L.

SUMMARYAn invertase was purified From Cichorium intyhbus L. roots by a combination of ammonium sulphate precipitation. Concanavalimn A affinity chromatography, anion exchange chromatography and gel filtration. A 230–fold purification and a final specific activity of 2.7U per mg protein was obtained. The enzyme has a M, of 93000 as estimated by gel filtration ans after SDS‐PAGE a single bannd with a M, of 50000 was found. Optima activity was found between pH 5 and pH 5.5.At low substrate concentrations equal amounts of fructose and glucose were produced (apperant Kw 55 mm). At higher sucrose concentrations progressively more isokestose was produced and fructose synthesis was inhibited. The high sucrose concentrations needed for isokestose systhesis do not support a physiological role for the enzyme in fructan biosynthesis.

The kinetic analysis of fructan biosynthesis in excised leaves of Lolium temulentum L.

SUMMARYExcised leaves of Lolium temulentum L. were fed 1CO. at 99% isotopic abundance in order to study the kinetics of incorporation of photosynthetically fixed carbon into different fructans and to determine the pathways of synthesis between sucrose and high‐molecular‐weight fructan. Following 6 h exposure to CO2 of detached and illuminated leaves, analysis of the accumulation of I‐keatose, 6–kestose and neokestose, and of the pattern of labelling observed in these isomers, suggested that they were each formed directly from sucrose at different rates. Similarly, the data suggested thai the tetrasaccharide isomers were formed directly from their trisaccharide precursors. Changes in the concentration of 13C both in the total water‐soluble component of the leaves and the fructan pool after 20h exposure to 13Co2 suggested that there was a continual turnover of frunctosyl residues. The formation of trisaccharides From sucrose and subsequent frunctosyl transfer to oligo‐ and polysaccharides of progressively higher molecular weight was consistent with a direct precursor product relationship. The stable concenttation of sucrose observed in leaves between 14 h and 48 h after excision indicated that fructan metabolism in L. temulentum may serve to regulate the sucrose content of the tissue.

Evidence for the de novo synthesis of Fructan by enzymes from higher plants: a Reappraisal of the SST/FFT model

SUMMARYFor the following reasons, it is argued that the enzymological evidnce is insufficient to sustain the SST/FFT model for fructan systhesis. (i) The original model was speculative. It was formulated without in vitro evidence for the synthesis of inulin of degree of polymerization greater than 3 (DP &gt;3), and without published evidence for the existence of a key enzyme (SST). The model is shown to be untestable in vitro. Subsequently, studies have not domonstrated the synthesis of inulin in vitro, except for trisaccharide. (ii) Most data cited in support of the model are concerned with the synthesis of the smallest fructans (trisaccharides) by crude enzyme extracts. Much of these data can be explained in terms of artifactual fructosyl trasfer by invertase, a common contaminant of plant enzyme preparations. (iii) There are few reports of the de novo enzymatic synthesis of fructan DP &gt;3 in plants. Where it has been reported, it was eatalyzed by crude or partially purified enzymes. Hence the number and properties of the enzymes involved is not known, providing no evidence for distinct SST and FFT enzymes. (iv) Assays for FFT require preformed fructan substates. FFT transfers fructose between preformed fructan molecules without net synthesis of fructan. Hence measurement of FFT is not evidence for net fructan synthesis. (v) Invertase can mimic the FFT reaction. (vi) The enzymes of fructan biosynthesis have not been purified and characterized in any plant species. Where functional separation of SST and FFT activities has been achieved. Reconstitution experiments have not been performed demostrating de novo fructan synthesis by the recombined activities. (vi) The SST/FFT model explains the synthesis of the linear β‐2,1‐linked homologous series of fructans. The model is inadequate to explain the synthesis of other linkage types and branched fructans. (vii) There is no demonstration of the enzymatic de novo synthesis of the full complement of tissue fructan by SST/FFT or otherwise, for any plant species.Although were is a substantial body of evidence which is consistent with the SST/FFT model, it is concluded that the hypothesis is flawed and that the enzymological data from all species do not validate the model. Enzyme systems from grasses are described which overcome some of these difficulties.

A reconsideration of fructan biosynthesis in storage roots of Asparagus officinalis L.*

summaryTotal soluble neutral carbohydrate (SNC) from storage roots of seven cultivars of Asparagus officinalis L. was analyzed by HPLC and TLC. Fructan accounted for roughly 90% of the total SNC and, of the fructan fraction, 81–98% was of degree of polymerization of five and larger (DP 5). Smaller oligosaccharides and sucrose represented a correspondingly small proportion of the total SNC. This observation differs from the generally accepted view of Asparagus fructan, which emphasizes the presence of small oligofructan in root tissue.On the basis of chromatographic mobility, neokestose and isokestose were identified as the two components of the trisaccharide fraction. Kestose did not accumulate in any of the cultivars studied.A crude enzyme extract of Asparagus roots was prepared using established methods involving dialysis for 5 d. Microbiological analysis of this preparation revealed heavy bacterial contamination. Oligofructan apparently persisted in this protein preparation despite the long period of dialysis. These observations have consequences for the interpretation of previous reports of in vitro fructosyl transferase data for Asparagus root enzymes. The validity of long‐term dialysis in the study of fructan enzymes is questioned.Crude protein extracts of roots were also desalted using a rapid gel‐exclusion method. When incubated with sucrose under conditions similar to established techniques, both invertase and fructosyl transferase activities were detected in this preparation. The trisaccharide products of the fructosyl transferase reaction were identified on TLC as neokestose, isokestose and kestose; the last did not accumulate in vivo. Oligofructans of DP &gt; 3 were also synthesized by this preparation and the pattern of products revealed by TLC was similar to previous reports. These oligofructan products of enzyme reactions were compared directly with the complete range of fructan structures accumulating in the tissue, using TLC. The enzyme products bore little resemblance to the native fructan pattern. Such enzyme activities are of doubtful physiological relevance and may be the result of artifactual or ancillary activities of other enzymes such as invertase.These findings are discussed with respect to previous studies of Asparagus root carbohydrates and serve as a basis for a critical reassessment of the enzymology of fructan biosynthesis both in this and other plant tissues.

Fructan biosynthesis in excised leaves of Lolium temulentum L.: IV. Cell-free 14 C labelling of specific oligofructans at low sucrose concentration.

Analyses of the kinetic properties and products of fructosyl transferases from Lolium temulentum L. suggest that these enzymes are artifacts of conventional extinction and/or assay methods. An alternative approach to the investigation of fructan biosynthesis in cell-free extracts has been developed. This approach employs the Sensitivity of radiotraeers to overcome the limitations of conventional quantitative methods for the analysis of fructan. Homogenates of leaves were prepared, which contained enzyme protein, endogenous sucrose and fructan. These extracts catalysed the rapid and efficient incorporation of radioactivity from [U-14 C]sucrose into material with a decree of polymerization greater than 2. The chromatographic pattern of these labelled products was identical to the pattern of native leaf fructan. Kestose, which does not accumulate to high concentrations in vivo but was a major product of our previous enzyme assay procedures, was not a product of these homogenates. The flux of radioactivity into oligofructan was sufficient to account for observed rates of fructan accumulation in the leaf And fructosyl transfer was observed at sucrose concentrations between 15 and 30 mol m-3 , which is within the probable physiological range of substrate concentration for L. temulentum leaf cells. Extracts derived from leaves which were not actively accumulating fructan were unable to label oligofructan. Labelling of fructan was not due to non-enzymic isotope exchange. Leal homogenates contained invertase and fructan hydrolase activities. Invertase activity was inhibited by pyridoxal HCl and pyridoxine MCI when these compounds were included in reactions at 20 mol m-3 . Inhibition of invertase activity was correlated with a marked stimulation of fructosyl transfer, suggesting competition for substrate. Because this system functions at physiologically relevant sucrose concentrations and produces a pattern of labelled products identical to native leaf fructan, we suggest that it represents a more convincing starting point for studies of the enzymology of fructan biosynthesis than previous cell-free extracts.

Fructan biosynthesis in excised leaves of Lolium temulentum L. III. A comparison of the in vitro properties of fructosyl transferase activities with the characteristics of in vivo fructan accumulation*

SUMMARYThe induction of fructan biosynthesis in excised, illuminated leaves of Lolium temulentum L. is accompanied by a concomitant increase in extractable sucrose: sucrose fructosyl transferase (SST) activity. In leaves treated with the L‐isomer of 2‐(4,methyl‐2,6,‐dinitroanilino)‐N‐methyl propionamide (L‐MDMP) fructan biosynthesis in vivo occurs in the absence of such an increase in extractable SST. suggesting that the activity measured in vitro is not an obligatory component of fructan biosynthesis in vivo.Sucrose:sucrose fructosyl transferase preparations from L. temulentum catalyse the formation of isokestose and kestose from sucrose. The rates of synthesis of these trisaccharides were determined in the range of sucrose concentration 10‐700 mol m−3. Increase in total trisaccharide synthesis was linear with respect to substrate concentration. Isokestose was Formed over the entire range whilst kestose synthesis was inactive below 8O mol m−3. Neither isokestose nor kestose synthesis was saturated at 700 mol m −3. The Km for isokestose synthesis was high at 380 mol m−4. Conventional kinetic analysis of kestose data failed, indicating that this activity does not obey classical Michaelis‐Menten kinetics.The intracellular sucrose concentration in leaf tissue was estimated to be 100 mol m−3. At this substrate concentration, isokestose synthesis would be Functioning well below the measured Km and kestose synthesis would be operating at the extreme lower limit of its activity range.Higher oligosaccharide products of fructosyl transferase incubations were not representative of the oligofructans which accumulate in the leaf. This data casts further doubt on the physiological significance of the measured fructosyl transferase activities.Isokestose synthesis was localized in vacuoles derived from leaf mesophyll protoplasts, confirming current theories of fructan biosynthesis. However, in view of the anomalous characteristics of fructosyl transferases in vitro, we propose that unambiguous assignment of the subcellular compartmentation of fructan metabolism in leaves of Lolium temulentum should await a fuller understanding of the enzymology of fructan biosynthesis in this species.

Fructan biosynthesis in excised leaves of Lolium temulentum L.

SUMMARYFructan oligosaccharides with degree of polymerization of 3–12 extracted from leaves of Lolium temulemtum L. were analyzed by normal‐phase thin‐layer chromatography. The pattern of oligosaccharides was more complex than that observed in tubers of Helianthus tuberosus L. and stem internodes of Hordeum vulgare L., indicating that fructans of multiple linkage types were present in this tissue.On the basis of co‐chromatography with known standards and constituent hexose analysis, two trisaccharides, neokestose (6‐G fructosyl sucrose) and isokestose (1‐F fructosyl sucrose) were found to accumulate to high concentrations. A third trisaccharide, thought to be kestose (6‐F fructosyl sucrose) was present at much lower tissue concentrations. An abundant tetrasaccharide with a chromatographic mobility similar to that of nystose (1‐F difructosyl sucrose) also accumulated.When carbohydrate‐depleted leaves were excised and continuously illuminated, total water‐soluble carbohydrate accumulation was linear with respect to time at a rate of 1–8 mg g‐1 fresh mass over a 30 h period. In the first 6h sucrose accumulated to lOmgg1, at which time fructan biosynthesis was detected. Isokestose appeared first, followed by neokestose and subsequently kestose and longer oligo‐ and polyfructosides. Changes in the concentrations of these compounds, and their radioactivity following feeding of 14CO2, was consistent with precursor‐product relationships between sucrose and trisaccharides, and between trisaccharides and oligo‐ and polyfructans.

Fructan biosynthesis in excised leaves of Lolium temulentum L.

SUMMARYFollowing the initiation of soluble carbohydrate accumulation in excised illuminated leaves of Lolium temulentum L., extractable sucrose: sucrose fructosyl transferase activity rose concomitant with the onset of fructan synthesis Extractable enzyme activity was sufficient to account for rates of fructan accumulation in vivo The major trisaccharide products of transferase activity in vitro were isokestose and kestose. This differed from the patterns of trisaccharide accumulation in vivo, where isokestose and neokestose predominated.Excised leaves treated with inhibitors of protein synthesis and of RNA metabolism accumulated water‐soluble carbohydrate at similar rates to control tissue, but did not convert sucrose into fructan. Inhibitor treatment also prevented increase in extractable sucrose: sucrose fructosyl transferase activity, suggesting that initiation of fructan biosynthesis was mediated by altered patterns of gene expression. These results are discussed in relation to the control of the synthesis of the oligofructosides characteristic of this tissue.

Extraction and characterization of the enzymes of fructan biosynthesis in timothy (Phleum pratense)

A preparation of phlein sucrase from seedling shoots of timothy (Phleum pratense L.) is described which catalyzed the synthesis of fructan with a mean molecular size of 34 000 using sucrose as the substrate. Activity was fully sedimentable at 25 000 × g, had a pH optimum of 7.0, and a Km for sucrose of 0.15 M. Activity was inhibited by β-mercaptoethanol and sodium diethyl dithiocarbamate. Raffinose and stachyose, but not members of the kestose series of oligofructans, could act as fructosyl donors in addition to sucrose. Formation of oligosaccharides during high molecular weight fructan synthesis was minimal, with synthesis occurring by a mechanism apparently analogous to bacterial levansucrase. These observations are discussed in relation to the in vivo patterns of fructan biosynthesis observed in different species of higher plants.

SUCROSE ACCUMULATION AND THE INITIATION OF FRUCTAN BIOSYNTHESIS IN LOLIUM TEMULENTUM L.

SummaryPlants of Lolium temulentum L. grown at 20 °C were transferred to 5 °C at the beginning of the light period. Sucrose accumulation was much more marked at low temperatures, with increases in rate detectable about 1 h following transfer. Exposure to low temperature over 7 d resulted in appearance of fructan, initially as oligosaccharides, and eventually as polymers of higher molecular weight. Sucrose accumulation was most marked in the blades of mature leaves, whereas the accumulation of fructan was maximal in the meristernatic regions of developing leaves. Chilling also led to a rise in activity of sucrose: sucrose fructosyltransferase, the first enzyme specifically involved in the biosynthesis of fructans. This activity was also higher in leaf meristems than in other regions of the plant.