Fructan 6G-fructosyltransferase Research Articles

In silico Exploration of Interaction Profile of Fructan 6G-Fructosyltransferase (6G-FTT) Enzyme with its Substrate 1-Kestose From Cichorium Intybus in Order to Determine Active Site of this Conjugation

Abstract: Chicory, or ‘Cichorium intybus’, is an erect perennial herb that can be used to treat a variety of diseases, including enteric sickness, diabetes, haemorrhoids, malaria, allergies, digestive issues, and more. The enzyme, called 6G-FFT, uses 1-kestose as a substrate for the synthesis of the more complex and branched fructans. To understand the residues involved in this conjugation reaction between 6G-FFT and 1-kestose, molecular docking study was performed. The amino acids ASP157, ASP33, SER32, TRP57, GLU211, ASP244, and GLU117 of the 6G-FFT protein showed a good interaction with the ligand by SP (Standard Precision) docking studies. Results for XP (Extra Precision) docking studies also suggest the amino acids THR153, GLU117, ASP244, GLU211, TRP57, ASP33 and ASP157 interact with the docking score of -10.6 Kcal/mol. Among these interacting amino acids, GLU117, ASP244, GLU211, TRP57, ASP33, and ASP157 were found to be common in both methods. This in silico study will be beneficial for further exploring conjugation reactions in Cichorium intybus.

Integrated Transcriptomics and Metabolomics Analysis of the Fructan Metabolism Response to Low-Temperature Stress in Garlic.

As the main reserve carbohydrate in garlic, fructan contributes to garlic's yield and quality formation. Numerous studies have shown that plant fructan metabolism induces a stress response to adverse environments. However, the transcriptional regulation mechanism of garlic fructan in low-temperature environments is still unknown. In this study, the fructan metabolism of garlic seedlings under low-temperature stress was revealed by transcriptome and metabolome approaches. With the extension of stress time, the number of differentially expressed genes and metabolites increased. Using weighted gene co-expression network analysis (WGCNA), three key enzyme genes related to fructan metabolism were screened (a total of 12 transcripts): sucrose: sucrose 1-fructosyltransferase (1-SST) gene; fructan: fructan 6G fructosyltransferase (6G-FFT) gene; and fructan 1-exohydrolase (1-FEH) gene. Finally, two hub genes were obtained, namely Cluster-4573.161559 (6G-FFT) and Cluster-4573.153574 (1-FEH). The correlation network and metabolic heat map analysis between fructan genes and carbohydrate metabolites indicate that the expression of key enzyme genes in fructan metabolism plays a positive promoting role in the fructan response to low temperatures in garlic. The number of genes associated with the key enzyme of fructan metabolism in trehalose 6-phosphate was the highest, and the accumulation of trehalose 6-phosphate content may mainly depend on the key enzyme genes of fructan metabolism rather than the enzyme genes in its own synthesis pathway. This study not only obtained the key genes of fructan metabolism in garlic seedlings responding to low temperatures but also preliminarily analyzed its regulatory mechanism, providing an important theoretical basis for further elucidating the cold resistance mechanism of garlic fructan metabolism.

Decreased expression of fructosyltransferase genes in asparagus roots may contribute to efficient fructan degradation during asparagus spear harvesting

Asparagus (Asparagus officinalis L.) accumulates inulin and inulin neoseries-type fructans in root, which are synthesized by three fructosyltransferases-sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100), and fructan:fructan 6G-fructosyltransferase (6G-FFT, EC 2.4.1.243). Fructans in roots are considered as energy sources for emerging of spears, and it has been demonstrated that a gradual decrease in root fructan content occurs during the spear harvesting season (budding and shooting up period). However, the roles of certain three fructosyltransferases during the harvest season have not yet been elucidated. Here, we investigated the variation in enzymatic activities and gene expression levels of three fructosyltransferases and examined sugar contents in roots before and during the spear harvest period. Two cDNAs, aoft2 and aoft3, were isolated from the cDNA library of roots. The respective recombinant proteins (rAoFT2 and rAoFT3), produced by Pichia pastoris, were characterized: rAoFT2 showed 1-FFT activity (producing nystose from 1-kestose), whereas rAoFT3 showed 1-SST activity (producing 1-kestose from sucrose). These reaction profiles of recombinant proteins were similar to those of native enzymes purified previously. These results indicate that aoft2 and aoft3 encoding 1-FFT and 1-SST are involved in fructan synthesis in roots. A gradual downregulation of fructosyltransferase genes and activity of respective enzymes was observed in roots during the harvest period, which also coincided with the decrease in fructooligosaccharides and increase in fructose due to fructan exohydrolase activity. These findings suggest that downregulation of fructosyltransferases genes during harvest time may contribute to efficient degradation of fructan required for the emergence of spears.

Fructans Are Differentially Distributed in Root Tissues of Asparagus.

Inulin- and neoseries-type fructans [fructooligosaccharides (FOS) and fructopolysaccharides] accumulate in storage roots of asparagus (Asparagus officinalis L.), which continue to grow throughout the lifespan of this perennial plant. However, little is known about the storage of fructans at the spatial level in planta, and the degree of control by the plant is largely uncertain. We have utilized mass spectrometry imaging (MSI) to resolve FOS distribution patterns in asparagus roots (inner, middle, and outer tissues). Fructan and proteome profiling were further applied to validate the differential abundance of various fructan structures and to correlate observed tissue-specific metabolite patterns with the abundance of related fructan biosynthesis enzymes. Our data revealed an increased abundance of FOS with higher degree of polymerization (DP > 5) and of fructopolysaccharides (DP11 to DP17) towards the inner root tissues. Three isoforms of fructan:fructan 6G-fructosyltransferase (6G-FFT), forming 6G-kestose with a β (2–6) linkage using sucrose as receptor and 1-kestose as donor, were similarly detected in all three root tissues. In contrast, one ß-fructofuranosidase, which likely exhibits fructan:fructan 1-fructosyltransferase (1-FFT) activity, showed very high abundance in the inner tissues and lower levels in the outer tissues. We concluded a tight induction of the biosynthesis of fructans with DP > 5, following a gradient from the outer root cortex to the inner vascular tissues, which also correlates with high levels of sucrose metabolism in inner tissues, observed in our study.

Purification and Characterization of a bifunctional fructan: fructan 6G-fructosyl transferase from garlic (Allium sativum)

A fructosyl transferase from garlic, fructan: fructan 6G-fructosyl transferase (6G-FFT), was purified and further characterized. 6G-FFT is a dimer glycoprotein around 120 kDa. Optimal activity was found at pH 6.0 and 25°C, and the enzyme was liable to heat instability and was inhibited by Ag+, Zn2+ and Cu2+. 6G-FFT is a bifunctional enzyme which has transferase and hydrolase activities. Transferase activity did not fit well the Michaelis-Menten kinetics, and its Hill coefficient (2.1±0.3) shows positive cooperativity of multiple binding sites for its substrate in the enzyme. Hydrolase activity shows similar Vmax and Km with transferase activity, which suggested that the substrate concentration must be high enough to offset the expense of hydrolysis and start the fructosyl-transferring activity. This bifunctional enzyme only recognized fructooligosaccharides with over two monosaccharide units as their substrates. These findings will provide a theoretical foundation to regulate the metabolism of fructans to prolong the storage of garlic.

Association of Candidate Genes With Submergence Response in Perennial Ryegrass.

Perennial ryegrass is a popular cool-season grass species due to its high quality for forage and turf. The objective of this study was to identify associations of candidate genes with growth and physiological traits to submergence stress and recovery after de-submergence in a global collection of 94 perennial ryegrass accessions. Accessions varied largely in leaf color, plant height (HT), leaf fresh weight (LFW), leaf dry weight (LDW), and chlorophyll fluorescence (Fv/Fm) at 7 days of submergence and in HT, LFW and LDW at 7 days of recovery in two experiments. Among 26 candidate genes tested by various models, single nucleotide polymorphisms (SNPs) in 10 genes showed significant associations with traits including 16 associations for control, 10 for submergence, and 8 for recovery. Under submergence, Lp1-SST encoding sucrose:sucrose 1-fructosyltransferase and LpGA20ox encoding gibberellin 20-oxidase were associated with LFW and LDW, and LpACO1 encoding 1-aminocyclopropane-1-carboxylic acid oxidase was associated with LFW. Associations between Lp1-SST and HT, Lp6G-FFT encoding fructan:fructan 6G-fructosyltransferase and Fv/Fm, LpCAT encoding catalase and HT were also detected under submergence stress. Upon de-submergence, Lp1-SST, Lp6G-FFT, and LpPIP1 encoding plasma membrane intrinsic protein type 1 were associated with LFW or LDW, while LpCBF1b encoding C-repeat binding factor were associated with HT. Nine significant SNPs in Lp1-SST, Lp6G-FFT, LpCAT, and LpACO1 resulted in amino acid changes with five substitutions found in Lp1-SST under submergence or recovery. The results indicated that allelic diversity in genes involved in carbohydrate and antioxidant metabolism, ethylene and gibberellin biosynthesis, and transcript factor could contribute to growth variations in perennial ryegrass under submergence stress and recovery after de-submergence.

Factors affecting fructosyltransferases and fructan exohydrolase activities in Agave tequilana Weber var. azul

There is great interest in the fructosyltransferases (FTFs) involved in fructan metabolism and agents affecting their activity. Agaves accumulate fructans, fructose polymers linked by glycosidic β(2–1) and β(2–6) bonds in linear or branched configurations. In plants, fructans provide protection under stress conditions. The sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT), fructan:fructan 6G-fructosyltransferase (6G-FFT), and fructan exohydrolase (FEH) activities were analyzed in micropropagated Agave tequilana plants in the absence and presence of HgCl2, AgNO3, MgCl2, sodium deoxycholate (DNa), and sodium dodecyl sulfate (SDS). Kestose, nystose and neokestose were synthesized by the respective FTFs. HgCl2 and AgNO3 inhibited all FTFs, mainly up to 90 % in 1-SST and 1-FFT. DNa increased 1-SST (32 %) and 1-FFT (45 %) activities, and SDS increased 6G-FFT activity by 96 %. Finally, AgNO3 inhibited FEH activity by 78 %. Our results might be relevant on the regulation of FTFs in agave and other crops, for instance by the increment the fructans synthesis in stressed plants.

Transcript profiling of fructan biosynthetic pathway genes reveals association of a specific fructosyltransferase isoform with the high sugar trait in Lolium perenne

Lolium perenne cultivars with elevated levels of fructans in leaf blades (high sugar-content grasses) have been developed to improve animal nutrition and reduce adverse environmental impacts of pastoral agricultural systems. Expression of the high sugar trait can vary substantially depending on genotype×environment (G×E) interactions. We grew three potential high sugar-content and a control cultivar in three temperature regimes and quantified water soluble carbohydrates (WSCs) and the expression of all functionally characterised L. perenne fructan pathway genes in leaf tissues. We also analysed the distribution, expression and sequence variation of two specific isoforms of Lp6G-FFT (fructan: fructan 6G-fructosyltransferase). Our study confirmed a significant G×E interaction affecting the accumulation of fructans in the high sugar-content cultivar AberDart, which accumulated higher levels of high DP (degree of polymerisation) fructans in blades compared to the control cultivar only when grown at 20°C (day)/10°C (night) temperatures. The cultivar Expo on the other hand accumulated significantly higher levels of high DP fructans in blades independent of temperature. Fructan levels in pseudostems were higher than in blades, and they increased markedly with decreasing temperature, but there was no consistent effect of cultivar in this tissue. The expression of the high sugar trait was generally positively correlated with transcript levels of fructosyltransferases. Presence and expression of only one of the two known 6G-FFT isoforms was positively correlated with high fructan biosynthesis, while the second isoform was associated with low fructan concentrations and positively correlated with fructan exohydrolase gene expression. The presence of distinct 6G-FFT sequence variants appears to be associated with the capacity of high sugar-content grasses to accumulate higher fructan levels particularly at warmer temperatures. These findings might be exploited for the selection and breeding of 'warm-effective' high sugar-content grasses to overcome some of the limitations of current high sugar-content ryegrass cultivars.

Fructan Metabolism in Developing Wheat (Triticum aestivum L.) Kernels

Although fructans play a crucial role in wheat kernel development, their metabolism during kernel maturation is far from being understood. In this study, all major fructan-metabolizing enzymes together with fructan content, fructan degree of polymerization and the presence of fructan oligosaccharides were examined in developing wheat kernels (Triticum aestivum L. var. Homeros) from anthesis until maturity. Fructan accumulation occurred mainly in the first 2 weeks after anthesis, and a maximal fructan concentration of 2.5 ± 0.3 mg fructan per kernel was reached at 16 days after anthesis (DAA). Fructan synthesis was catalyzed by 1-SST (sucrose:sucrose 1-fructosyltransferase) and 6-SFT (sucrose:fructan 6-fructosyltransferase), and to a lesser extent by 1-FFT (fructan:fructan 1-fructosyltransferase). Despite the presence of 6G-kestotriose in wheat kernel extracts, the measured 6G-FFT (fructan:fructan 6G-fructosyltransferase) activity levels were low. During kernel filling, which lasted from 2 to 6 weeks after anthesis, kernel fructan content decreased from 2.5 ± 0.3 to 1.31 ± 0.12 mg fructan per kernel (42 DAA) and the average fructan degree of polymerization decreased from 7.3 ± 0.4 (14 DAA) to 4.4 ± 0.1 (42 DAA). FEH (fructan exohydrolase) reached maximal activity between 20 and 28 DAA. No fructan-metabolizing enzyme activities were registered during the final phase of kernel maturation, and fructan content and structure remained unchanged. This study provides insight into the complex metabolism of fructans during wheat kernel development and relates fructan turnover to the general phases of kernel development.

Towards a better understanding of the generation of fructan structure diversity in plants: molecular and functional characterization of a sucrose:fructan 6-fructosyltransferase (6-SFT) cDNA from perennial ryegrass (Lolium perenne).

The main storage compounds in Lolium perenne are fructans with prevailing β(2–6) linkages. A cDNA library of L. perenne was screened using Poa secunda sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. A full-length Lp6-SFT clone was isolated as shown by heterologous expression in Pichia pastoris. High levels of Lp6-SFT transcription were found in the growth zone of elongating leaves and in mature leaf sheaths where fructans are synthesized. Upon fructan synthesis induction, Lp6-SFT transcription was high in mature leaf blades but with no concomitant accumulation of fructans. In vitro studies with the recombinant Lp6-SFT protein showed that both 1-kestotriose and 6G-kestotriose acted as fructosyl acceptors, producing 1- and 6-kestotetraose (bifurcose) and 6G,6-kestotetraose, respectively. Interestingly, bifurcose formation ceased and 6G,6-kestotetraose was formed instead, when recombinant fructan:fructan 6G-fructosyltransferase (6G-FFT) of L. perenne was introduced in the enzyme assay with sucrose and 1-kestotriose as substrates. The remarkable absence of bifurcose in L. perenne tissues might be explained by a higher affinity of 6G-FFT, as compared with 6-SFT, for 1-kestotriose, which is the first fructan formed. Surprisingly, recombinant 6-SFT from Hordeum vulgare, a plant devoid of fructans with internal glucosyl residues, also produced 6G,6-kestotetraose from sucrose and 6G-kestotriose. In the presence of recombinant L. perenne 6G-FFT, it produced 6G,6-kestotetraose from 1-kestotriose and sucrose, like L. perenne 6-SFT. Thus, we demonstrate that the two 6-SFTs have close catalytic properties and that the distinct fructans formed in L. perenne and H. vulgare can be explained by the presence of 6G-FFT activity in L. perenne and its absence in H. vulgare.

Transforming a fructan:fructan 6G-fructosyltransferase from perennial ryegrass into a sucrose:sucrose 1-fructosyltransferase.

Fructosyltransferases (FTs) synthesize fructans, fructose polymers accumulating in economically important cool-season grasses and cereals. FTs might be crucial for plant survival under stress conditions in species in which fructans represent the major form of reserve carbohydrate, such as perennial ryegrass (Lolium perenne). Two FT types can be distinguished: those using sucrose (S-type enzymes: sucrose:sucrose 1-fructosyltransferase [1-SST], sucrose:fructan 6-fructosyltransferase) and those using fructans (F-type enzymes: fructan:fructan 1-fructosyltransferase [1-FFT], fructan:fructan 6G-fructosyltransferase [6G-FFT]) as preferential donor substrate. Here, we report, to our knowledge for the first time, the transformation of an F-type enzyme (6G-FFT/1-FFT) into an S-type enzyme (1-SST) using perennial ryegrass 6G-FFT/1-FFT (Lp6G-FFT/1-FFT) and 1-SST (Lp1-SST) as model enzymes. This transformation was accomplished by mutating three amino acids (N340D, W343R, and S415N) in the vicinity of the active site of Lp6G-FFT/1-FFT. In addition, effects of each amino acid mutation alone or in combination have been studied. Our results strongly suggest that the amino acid at position 343 (tryptophan or arginine) can greatly determine the donor substrate characteristics by influencing the position of the amino acid at position 340. Moreover, the presence of arginine-343 negatively affects the formation of neofructan-type linkages. The results are compared with recent findings on donor substrate selectivity within the group of plant cell wall invertases and fructan exohydrolases. Taken together, these insights contribute to our knowledge of structure/function relationships within plant family 32 glycosyl hydrolases and open the way to the production of tailor-made fructans on a larger scale.

Food Biochemical Study on Fructans and Related Synthesis Enzymes

There are more than 500 kinds of oligosaccharide occurring in nature, and all of these are synthesized by chemical and enzymatic reactions. Various studies have recently identified physiological and physical applications of such oligosaccharides in agricultural chemistry, nutrition and medicine. New technologies for the production of oligosaccharides from natural resources have been developed in the food industry and several oligosaccharides are now produced on a large scale as ingredients in animal feed. We have investigated the isolation of fructo-oligosaccharides, as well as the enzymatic synthesis of oligosaccharides that have functional activities as tertiary functional ingredients in food. We have studied the purification and characterization of several fructosyltransferases from asparagus roots and onion bulbs, including sucrose: sucrose 1-fructosyltransferase (1-SST), fructan: fructan 1-fructosyltransferase (1-FFT), and the novel enzyme fructan:fructan 6G-fructosyltransferase (6G-FFT). We have also reported that asparagus 1-FFT synthesized new functional oligosaccharides elongated with one or two additional fructose units by fructosyltransfer from 1-kestose to 4G-β-D-galactosylsucrose, and this compound selectively stimulates the growth of Bifidobacteria. For industrial applications, we attempted the isolation and expression of cDNAs encoding 6G-FFT, 1-FFT and 1-SST from asparagus plants. The cDNAs encoding 6G-FFT, 1-FFT and 1-SST were isolated from a cDNA library of asparagus leaves or roots, and the isolated cDNAs were designated aoft1, aoft2 and aoft3, respectively. The deduced amino acid sequences of these cDNAs showed high homology with those of plant fructosyltransferases. Expression of these cDNAs was performed using Pichia pastoris. The recombinant protein from Pichia transformed with aoft1 produced 1F,6G-di-β-D-fructofuranosylsucrose, neokestose and sucrose from 1-kestose, while the transformant with an empty vector produced no saccharides. These results confirmed that 6G-FFT was expressed in P. pastoris. Similarly, the recombinant protein from Pichia transformed with aoft2 produced nystose from 1-kestose and the recombinant protein with aoft3 produced 1-kestose from sucrose. These results confirmed that 1-FFT and 1-SST were expressed in P. pastoris, and that the recombinant proteins had enzymatic properties similar to those of 6G-FFT, 1-FFT and 1-SST from asparagus roots. We then examined the conversion of substrate specificity from 6G-FFT to 1-FFT by point mutations in the β-fructosidase motif. The asparagine in this motif in aoft1 was changed to serine, and the mutant recombinant protein was characterized. We found that this amino acid substitution in wild-type aoft1 changed the substrate specificity from 6G-FFT to 1-FFT. Finally, we studied the activities of the main enzymes involved in the synthesis and hydrolysis of fructo-oligosaccharides during the post-harvest life of onion bulbs and asparagus spears, and discuss the mechanisms triggering these enzyme activities, as well as the mechanisms by which fructo-oligosaccharides contribute to the quality and perishability of the vegetables.

Improved fructan accumulation in perennial ryegrass transformed with the onion fructosyltransferase genes 1-SST and 6G-FFT

Carbohydrate limitation has been identified as a main cause of inefficient nitrogen use in ruminant animals, which feed mainly on fresh forage, hay and silage. This inefficiency results in suboptimal meat and milk productivity. One important molecular breeding strategy is to improve the nutritional value of ryegrass (Lolium perenne) by increasing the fructan content through expression of heterologous fructan biosynthetic genes. We developed perennial ryegrass lines expressing sucrose:sucrose 1-fructosyltransferase and fructan:fructan 6G-fructosyltransferase genes from onion (Allium cepa) which exhibited up to a 3-fold increased fructan content. Further, the high fructan content was stable during the growth period, whereas the fructan content in an elite variety, marketed as a high sugar variety, dropped rapidly after reaching its maximum and subsequently remained low.

Saccharide and Fructooligosaccharide Contents, and Invertase, 1-KHE, 1-SST, 1-FFT and 6G-FFT Activities in Green Asparagus Spears during Storage: Effects of Temperature and Spear Portion

Fresh spears of asparagus were stored in the dark at 4, 10 or 20°C for 2 weeks. During storage contents of glucose, fructose, sucrose, 1-kestose, neokestose and nystose, and activities of invertase, 1-kestose hydrolyzing enzyme (1-KHE), sucrose: sucrose 1-fructosyltransferase (1-SST), fructan: fructan 1-fructosyltransferase (1-FFT) and fructan: fructan 6G-fructosyltransferase (6G-FFT) were determined in the top, middle and bottom portions of the spears. A gradient was observed, from the bottom to the top, for glucose, fructose and sucrose which constitute the major proportion of carbohydrates, while fructooligosaccharides, neokestose and nystose, exhibited low levels. Glucose and fructose varied significantly during storage, while sucrose was stable. The average variations were from 7.8 to 12.21 mg/g FW in the middle portion and 7.88 to 13.52 mg/g FW in the bottom portion for glucose and fructose, respectively. 1-Kestose and nystose increased at the end of the storage period and this increase was more apparent at 20°C. Invertase activity showed similar variation at 4 and 10°C but increased sharply after 2 days, before decreasing abruptly after 1 week of storage, while 1-kestose hydrolyzing activity showed a similar pattern to that of invertase activity. 1-SST did not vary in the bottom portion but initially increased in the middle and top portions. 1-FFT was high in the top portion and decreased during storage, while in the middle and bottom portions its activity varied slightly. The variation of 6G-FFT activity was similar to that of 1-FFT, however, the level of 6G-FFT was higher, and the 6G-FFT to 1-FFT activity ratio was temperature independent. These results suggest that short fructooligosaccharides and their metabolizing enzymes could play a role of balance between the hydrolysis and synthesis activities of carbohydrates. The high content of sugars may also extend the rapid decline of sugars in the top portion of the spears.

Impact of defoliation frequency on regrowth and carbohydrate metabolism in contrasting varieties of Lolium perenne.

The aims of the study were to gain a better understanding of fructan metabolism regulation during regrowth of Lolium perenne, and to evaluate the role of fructans of remaining tissues as well as carbon assimilation of new leaf tissues in refoliation. Two varieties that contrast for carbohydrate metabolism, Aurora and Perma, were subject to severe and frequent or infrequent defoliations before regrowth. Aurora, which had a greater content of fructans in leaf sheaths than Perma before defoliation, produced more leaf biomass within the 4 days following the first cut. At the end of the regrowth period, Aurora produced more leaf biomass than Perma. Photosynthetic parameters, which were barely affected by defoliation frequency, could not explain these differences. Fructan synthesising activities [sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 6G-fructosyltransferase (6G-FFT)], declined after defoliation. In elongating leaf bases, corresponding transcript levels did not decline concomitantly, suggesting a post-transcriptional regulation of expression, while in leaf sheaths the gene expression pattern mostly followed the time-course of the enzyme activities. Regulation of Lp1-SST and Lp6G-FFT gene expression depends, therefore, on the sink-source status of the tissue after defoliation. During the phase of reserve accumulation, fructosyltransferase activities together with corresponding transcripts increased more in frequently defoliated plants than in infrequently defoliated plants.

Developing fructan‐synthesizing capability in a plant invertase via mutations in the sucrose‐binding box

Fructans are fructose polymers that are synthesized from sucrose by fructosyltransferases. Fructosyltransferases are present in unrelated plant families suggesting a polyphyletic origin for their transglycosylation activity. Based on sequence comparisons and enzymatic properties, fructosyltransferases are proposed to have evolved from vacuolar invertases. Between 1% and 5% of the total activity of vacuolar invertase is transglycosylating activity. We investigated the nature of the changes that can convert a hydrolysing invertase into a transglycosylating enzyme. Remarkably, replacing 33 amino acids (amino acids 143-175) corresponding to the N-terminus of the mature onion vacuolar invertase with the corresponding region of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) led to a shift in activity from hydrolysis of sucrose towards transglycosylation between two sucrose molecules. The substituted N-terminal region contains the sucrose-binding box that harbours the nucleophile involved in sucrose hydrolysis (Asp164). Subsequent research into the individual amino acids responsible for the enhanced transglycosylation activity revealed that mutations in amino acids Trp161 and Asn166, can give rise to a shift towards polymerase activity. Changing the amino acid at either of these positions in the sucrose-binding box increases the transglycosylation capacity of invertases two- to threefold compared to wild type. Combining the two mutations had an additive effect on transglycosylation ability, resulting in an approximately fourfold enhancement. The mutations generated correspond with natural variation present in the sucrose-binding boxes of vacuolar invertases and fructosyltransferases. These relatively small changes that increase the transglycosylation capacity of invertases might explain the polyphyletic origin of the fructan accumulation trait.

Molecular and functional characterization of a cDNA encoding fructan:fructan 6G-fructosyltransferase (6G-FFT)/fructan:fructan 1-fructosyltransferase (1-FFT) from perennial ryegrass (Lolium perenne L.)

Fructans are the main storage compound in Lolium perenne. To account for the prevailing neokestose-based fructan synthesis in this species, a cDNA library of L. perenne was screened by using the onion (Allium cepa) fructan:fructan 6G-fructosyltransferase (6G-FFT) as a probe. A full length Lp6G-FFT clone was isolated with significant homologies to vacuolar type fructosyltransferases and invertases. The functionality of the cDNA was tested by heterologous expression in Pichia pastoris. The recombinant protein demonstrated both 6G-FFT and fructan:fructan 1-fructosyltransferase activities (1-FFT) with a maximum 6G-FFT/1-FFT ratio of two. The activity of 6G-FFT was investigated with respect to developmental stage, tissue distribution, and alterations in carbohydrate status expression and compared to sucrose:sucrose 1-fructosyltransferase (1-SST). Lp6G-FFT and Lp1-SST were predominantly expressed in the basal part of elongating leaves and leaf sheaths. Expression of both genes declined along the leaf axis, in parallel with the spatial occurrence of fructan and fructosyltransferase activities. Surprisingly, Lp6G-FFT was highly expressed in photosynthetically active tissues where very low extractable fructosyltransferase activity and fructan amounts were detected, suggesting a post-transcriptional regulation of expression. Lp6G-FFT gene expression increased only in elongating leaves following similar increases of sucrose content in blades, sheaths, and elongating leaf bases. Regulation of Lp6G-FFT gene expression depends on the tissue according to its sink-source status.

Variation of Fructooligosaccharides and their Metabolizing Enzymes in Onion Bulb (Allium cepa L. cv. Tenshin) During Long-term Storage

ABSTRACT: The objective of this study was to assess the status of fructooligosaccharides (FOS) in onion bulbs (Allium cepa L. cv. Tenshin) and their metabolizing-enzymes—1-fructoexohydrolase (1-FEH), 1-kestose hydrolyzing enzyme (1-KH), fructan:fructan 1F-fructosyltransferase (1-FFT) and fructan:fructan 6G-fructosyltransferase (6G-FFT)—during storage at 15°C. Fructose varies slightly, whereas 1-kestose peaked after 6 wk and then decreased progressively during the last 18 wk of storage. Lower degree of polymerization (DP) 3 to 6) FOS, higher (DP 7 to 12) FOS, total FOS, and total carbohydrates showed similar and close patterns during 24 wk. They varied slightly at the beginning of the storage period; afterward they decreased progressively and regularly during the last 20 wk of storage. 1-FEH and 1-KH activities were low but peaked abruptly after 12 and 16 wk, respectively, after which they decreased to levels higher (1-FEH) or similar (1-KH) to those observed at the beginning of the storage. Surprisingly, 1-FFT activity showed similar pattern to the variation of 1-KH hydrolyzing activity; on the other hand, 6G-FFT, although higher, was stable during 16 wk but decreased after that. The results allowed us to associate FOS to the dormancy and sprouting states, and the peaks of the degrading enzymes were shown to signal the release of dormancy of onion bulb.

Fructooligosaccharides changes during maturation in inflorescences and seeds of onion (Allium cepa L. ‘W202’)

The distribution of fructooligosaccharides (FOS) and their related enzyme activities during the development of the inflorescences and the maturation of seeds in onion (Allium cepa L.) were investigated. Concentrations of glucose, fructose and sucrose in three inflorescence parts – complete floral parts (FP), pedicels (PD) and involucres (IV) - increased steadily during the first 8 wk of sampling and then decreased over for the next 6 wk. However the final glucose, fructose and sucrose contents were not significantly higher than those observed at the beginning of sampling. Surprisingly, FOS concentration in the inflorescence parts for the saccharides studied showed similar patterns with concentrations increasing during the first 6 wk and decreasing during the last 6 wk of the study. On the other hand, FL and PD parts contained higher amounts of FOS than IV parts. The activities of sucrose:sucrose 1-fructosyltransferase (SST), fructan: fructan 6G-fructosyltransferase (6G-FFT) and fructan:fructan 1F-fructosyltransferase (1-FFT) showed similar patterns in seeds, decreasing slowly during the first weeks and remaining stable thereafter. The ratio 6G-FFT to 1-FFT decreased after 9 wk from 2.40 to 1.44 with an average of 2.1. In contrast, activities of invertase and β-fructofuranosidase increased progressively, although β -fructofuranosidase activity was twice as high as invertase activity during the last 2 wk. The ratio of synthesizing to hydrolysing activities decreased progressively to 0.2 and was below the value 1 after 9 wk. Thus, it seems that seed maturation was reached during the last week of August, which is 2 to 3 wk earlier than seed harvest normally occurs in Hokkaido. Key words: Fructooligosaccharides, flower organs, seeds, onion

Using Natural Variation to Investigate the Function of Individual Amino Acids in the Sucrose-Binding Box of Fructan:Fructan 6G-Fructosyltransferase (6G-FFT) in Product Formation

Enzymes of the glycosyl hydrolase family 32 are highly similar with respect to primary sequence but catalyze divergent reactions. Previously, the importance of the conserved sucrose-binding box in determining product specificity of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) was established [Ritsema et al., 2004, Plant Mol. Biol. 54: 853-863]. Onion 6G-FFT synthesizes the complex fructan neo-series inulin by transferring fructose residues to either a terminal fructose or a terminal glucose residue. In the present study we have elucidated the molecular determinants of product specificity by substitution of individual amino acids of the sucrose binding box with amino acids that are present on homologous positions in other fructosyltransferases or vacuolar invertases. Substituting the presumed nucleophile Asp85 of the beta-fructosidase motif resulted in an inactive enzyme. 6G-FFT mutants S87N and S87D did not change substrate or product specificities, whereas mutants N84Y and N84G resulted in an inactive enzyme. Most interestingly, mutants N84S, N84A, and N84Q added fructose residues preferably to a terminal fructose and hardly to the terminal glucose. This resulted in the preferential production of inulin-type fructans. Combining mutations showed that amino acid 84 determines product specificity of 6G-FFT irrespective of the amino acid at position 87.