Comparative study of transgenic Brachypodium distachyon expressing sucrose:fructan 6-fructosyltransferases from wheat and timothy grass with different enzymatic properties

Fructans can act as cryoprotectants and contribute to freezing tolerance in plant species, such as in members of the grass subfamily Pooideae that includes Triticeae species and forage grasses. To elucidate the relationship of freezing tolerance, carbohydrate composition and degree of polymerization (DP) of fructans, we generated transgenic plants in the model grass species Brachypodium distachyon that expressed cDNAs for sucrose:fructan 6-fructosyltransferases (6-SFTs) with different enzymatic properties: one cDNA encoded PpFT1 from timothy grass (Phleum pratense), an enzyme that produces high-DP levans; a second cDNA encoded wft1 from wheat (Triticum aestivum), an enzyme that produces low-DP levans. Transgenic lines expressing PpFT1 and wft1 showed retarded growth; this effect was particularly notable in the PpFT1 transgenic lines. When grown at 22 °C, both types of transgenic line showed little or no accumulation of fructans. However, after a cold treatment, wft1 transgenic plants accumulated fructans with DP = 3–40, whereas PpFT1 transgenic plants accumulated fructans with higher DPs (20 to the separation limit). The different compositions of the accumulated fructans in the two types of transgenic line were correlated with the differences in the enzymatic properties of the overexpressed 6-SFTs. Transgenic lines expressing PpFT1 accumulated greater amounts of mono- and disaccharides than wild type and wft1 expressing lines. Examination of leaf blades showed that after cold acclimation, PpFT1 overexpression increased tolerance to freezing; by contrast, the freezing tolerance of the wft1 expressing lines was the same as that of wild type plants. These results provide new insights into the relationship of the composition of water-soluble carbohydrates and the DP of fructans to freezing tolerance in plants.     

Functional characterization of a sucrose:fructan 6-fructosyltransferase of the cold-resistant grass Bromus pictus by heterelogous expression in Pichia pastoris and Nicotiana tabacum and its involvement in freezing tolerance

We have previously reported the molecular characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) of Bromus pictus, a graminean species from Patagonia, tolerant to cold and drought. Here, this enzyme was functionally characterized by heterologous expression in Pichia pastoris and Nicotiana tabacum. Recombinant P. pastoris Bp6-SFT showed comparable characteristics to barley 6-SFT and an evident fructosyltransferase activity synthesizing bifurcose from sucrose and 1-kestotriose. Transgenic tobacco plants expressing Bp6-SFT, showed fructosyltransferase activity and fructan accumulation in leaves. Bp6-SFT plants exposed to freezing conditions showed a significantly lower electrolyte leakage in leaves compared to control plants, indicating less membrane damage. Concomitantly these transgenic plants resumed growth more rapidly than control ones. These results indicate that Bp6-SFT transgenic tobacco plants that accumulate fructan showed enhanced freezing tolerance compared to control plants.     

Engineering fructan metabolism in plants

Fructans, or polyfructosylsucroses, are storage carbohydrates present in many higher plants. They are also considered healthy food ingredients. Engineering crops into high level production of specific fructan molecules is one of the mayor strategic research goals. Understanding the properties of fructosyltransferases is important, in order to direct the synthesis of fructans. In plants at least two fructosyltransferases are needed to synthesise fructans. One enzyme synthesises the fructan trisaccharide 1-kestose, the next enzyme uses 1-kestose for elongation and/or modification, producing longer fructans. The specificity of fructosyltransferases determines the type of glycosidic bond formed and the donor and acceptor substrates used. This enables the synthesis of many structurally diverse fructans. The production of these molecules in crops such as sugar beet and potato makes the commercial use of fructans feasible.     

Production of tailor-made fructans in sugar beet by expression of onion fructosyltransferase genes

The consumption of fructans as a low caloric food ingredient or dietary fibre is rapidly increasing due to health benefits. Presently, the most important fructan source is chicory, but these fructans have a simple linear structure and are prone to degradation. Additional sources of high-quality tailor-made fructans would provide novel opportunities for their use as food ingredients. Sugar beet is a highly productive crop that does not normally synthesize fructans. We have introduced specific onion fructosyltransferases into sugar beet. This resulted in an efficient conversion of sucrose into complex, onion-type fructans, without the loss of storage carbohydrate content.     

Effect of ethylene glycol and glycerol fructosides on the activity and product specificity of bacterial and plant fructosyltransferases

Fructosyltransferases (FTs) are key enzymes in plants and bacteria to synthesize fructans. To gain insight on the specificity of the hexose subsites in the active site of FTs, ethylene glycol fructoside (EGF) and glycerol fructoside (GF), containing fructose in the furanose configuration, were synthesized in vitro and used as substrates to study the effect on the activity of bacterial levansucrase (BsLS), chicory root sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT). The results demonstrated that EGF and GF, at physiologically relevant concentrations, were efficient acceptor substrates for BsLS and 1-FFT, but not for 1-SST. EGF and GF cannot be used as donor substrates for BsLS, 1-SST and 1-FFT. A model is proposed to explain the subsite specificity differences between the three FTs involved in this study.     

Endogenous hormone concentrations correlate with fructan metabolism throughout the phenological cycle in Chrysolaena obovata

Background and Aims Chrysolaena obovata, an Asteraceae of the Brazilian Cerrado, presents seasonal growth, marked by senescence of aerial organs in winter and subsequent regrowth at the end of this season. The underground reserve organs, the rhizophores, accumulate inulin-type fructans, which are known to confer tolerance to drought and low temperature. Fructans and fructan-metabolizing enzymes show a characteristic spatial and temporal distribution in the rhizophores during the developmental cycle. Previous studies have shown correlations between abscisic acid (ABA) or indole acetic acid (IAA), fructans, dormancy and tolerance to drought and cold, but the signalling mechanism for the beginning of dormancy and sprouting in this species is still unknown.Methods Adult plants were sampled from the field across phenological phases including dormancy, sprouting and vegetative growth. Endogenous concentrations of ABA and IAA were determined by GC-MS-SIM (gas chromatography–mass spectrometry–selected ion monitoring), and measurements were made of fructan content and composition, and enzyme activities. The relative expression of corresponding genes during dormancy and sprouting were also determined.Key Results Plants showed a high fructan 1-exohydrolase (EC 3.2.1.153) activity and expression during sprouting in proximal segments of the rhizophores, indicating mobilization of fructan reserves, when ABA concentrations were relatively low and precipitation and temperature were at their minimum values. Concomitantly, higher IAA concentrations were consistent with the role of this regulator in promoting cell elongation and plant growth. With high rates of precipitation and high temperatures in summer, the fructan-synthesizing enzyme sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) showed higher activity and expression in distal segments of the rhizophores, which decreased over the course of the vegetative stage when ABA concentrations were higher, possibly signalling the entry into dormancy.Conclusions The results show that fructan metabolism correlates well with endogenous hormone concentrations and environmental changes, suggesting that the co-ordinated action of carbohydrate metabolism and hormone synthesis enables C. obovata to survive unfavourable field conditions. Endogenous hormone concentrations seem to be related to regulation of fructan metabolism and to the transition between phenophases, signalling for energy storage, reserve mobilization and accumulation of oligosaccharides as osmolytes.     

Low temperature and defoliation affect fructan-metabolizing enzymes in different regions of the rhizophores of Vernonia herbacea

In addition to the storage function, fructans in Asteraceae from floras with seasonal growth have been associated with drought and freezing tolerance. Vernonia herbacea, native of the Brazilian Cerrado, bears underground reserve organs, rhizophores, accumulating inulin-type fructans. The rhizophore is a cauline branched system with positive geotropic growth, with the apex (distal region) presenting younger tissues; sprouting of new shoots occurs by development of buds located on the opposite end (proximal region). Plants induced to sprouting by excision of the aerial organs present increased 1-fructan exohydrolase (1-FEH) activity in the proximal region, while plants at the vegetative stage present high 1-sucrose:sucrose fructosyltransferase (1-SST) in the distal region. The aim of the present study was to analyze how low temperature (5 °C) could affect fructan-metabolizing enzymes and fructan composition in the different regions of the rhizophores of intact and excised plants. 1-SST and 1-fructan:fructan fructosyltransferase (1-FFT) were higher in the distal region decreasing towards the proximal region in intact plants at the vegetative phase, and were drastically diminished when cold and/or excision were imposed. In contrast, 1-FEH increased in the proximal region of treated plants, mainly in excised plants subjected to cold. The ratio fructo-oligo to fructo-polysaccharides was significantly higher in plants exposed to low temperature (1.17 in intact plants and 1.64 in excised plants) than in plants exposed to natural temperature conditions (0.84 in intact vegetative plants and 0.58 in excised plants), suggesting that oligosaccharides are involved in the tolerance of plants to low temperature via 1-FEH, in addition to 1-FFT. Principal component analysis indicated different response mechanisms in fructan metabolism under defoliation and low temperature, which could be interpreted as part of the strategies to undergo unfavorable environmental conditions prevailing in the Cerrado during winter.     

Oligosaccharide intermediates of fructan synthesis in Lolium temulentum

Evidence is presented that fructan accumulation in leaves of Lolium temulentum plants grown at 5° proceeded via the synthesis of trisaccharide intermediates. Studies on the oligosaccharide components of this tissue indicated that the major intermediate was probably 1^F-fructosyl sucrose (isokestose) but that two distinct series of oligofructosides could be isolated. One of these had chromatographic properties identical to the 1,2-linked inulin series from Helianthus tuberosus. The relationship of this synthetic pattern to the structure of grass fructans and their accumulation in other species is discussed.     

Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance

Fructans are water-soluble fructose oligomers and polymers thatare based on sucrose, and have been implicated in protectingplants against water stress. Rice (Oryza sativa L.) is highlysensitive to chilling temperatures, and is not able to synthesizefructans. Two wheat fructan-synthesizing enzymes, sucrose:sucrose1-fructosyltransferase, encoded by wft2, or sucrose:fructan6-fructosyltransferase, encoded by wft1, were introduced intorice plants, and rice transformants that accumulate fructanswere successfully obtained. The mature leaf blades of transgenicrice lines with wft2 or wft1 accumulated 16.2 mg g^–1 FWof oligo- and polysaccharides mainly composed of inulin oligomersof more than DP7, and 3.7 mg g^–1 FW of oligo- and polysaccharides,mainly composed of phlein oligomers of more than DP15, respectively.The transgenic rice seedlings with wft2 accumulated significantlyhigher concentrations of oligo- and polysaccharides than non-transgenicrice seedlings, and exhibited enhanced chilling tolerance. Theoligo- and polysaccharide concentrations of seedlings expressingwft1 were obviously lower than those of lines expressing wft2,and no correlation between oligo- and polysaccharide concentrationsand chilling tolerance was detected in wft1-expressing ricelines. The results suggest that transgenic rice lines expressingwheat-derived fructosyltransferase genes accumulated large amountsof fructans in mature leaf blades and exhibited enhanced chillingtolerance at the seedling stage. This is the first report owingthat fructan accumulation enhanced tolerance to non-freezinglow temperatures. Key words: Chilling tolerance, fructan, fructosyltransferase, Oryza sativa, transgenic plant     

Influencing the binding configuration of sucrose in the active sites of chicory fructan 1-exohydrolase and sugar beet fructan 6-exohydrolase

The hydrolytic plant enzymes of family 32 of glycoside hydrolases (GH32), including acid cell wall type invertases (EC 3.2.1.26), fructan 1-exohydrolases (1-FEH; EC 3.2.1.153) and fructan 6-exohydrolases (6-FEH; EC 3.2.1.154), are very similar at the molecular and structural levels, but are clearly functionally different. The work presented here aims at understanding the evolution of enzyme specificity and functional diversity in this family by means of site-directed mutagenesis. It is demonstrated for the first time that invertase activity can be introduced in an S101L mutant of chicory (Cichorium intybus) 1-FEH IIa by influencing the orientation of Trp 82. At high sucrose and enzyme concentrations, a shift is proposed from a stable inhibitor configuration to an unstable substrate configuration. In the same way, invertase activity was introduced in Beta vulgaris 6-FEH by introducing an acidic amino acid in the vicinity of the acid-base catalyst (F233D mutant), creating a beta-fructofuranosidase type of enzyme with dual activity against sucrose and levan. As single amino acid substitutions can influence the donor substrate specificity of FEHs, it is predicted that plant invertases and FEHs may have diversified by introduction of a very limited number of mutations in the common ancestor.     

De-novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes (sucrose: sucrose 1-fructosyl transferase and fructan: fructan 1-fructosyl transferase) from chicory roots (Cichorium intybus L.)

Although fructans occur widely in several plant families and they have been a subject of investigation for decennia, the mechanism of their biosynthesis is not completely elucidated. We succeeded in purifying a fructan: fructan 1-fructosyl transferase (1-FFT; EC 2.4.1.100) from chicory roots (Cichorium intybus L. var. foliosum cv. Flash). In combination with the purified chicory root sucrose: sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99), this enzyme synthesized a range of naturally occurring chicory fructans (inulins) from sucrose as the sole substrate. Starting from physiologically relevant sucrose concentrations, inulins up to a degree of polymerization (DP) of about 20 were synthesized in vitro after 96 h at 0°C. Neither 1-SST, nor 1-FFT alone could mediate the observed fructan synthesis. Fructan synthesis in vitro was compared starting from 50, 100 and 200 mM sucrose, respectively. The initiation of (DP > 3)-fructan synthesis was found to be correlated with a certain ratio of 1 kestose to sucrose. The data presented now provide strong evidence to validate the 1-SST/1-FFT model for in-vivo fructan synthesis, at least in the Asteraceae.Abbreviations DP degree of polymerization - 1-FFT fructan: fructan 1-fructosyl transferase - 1-SST sucrose: sucrose 1-fructosyl transferase The authors thank E. Nackaerts for valuable technical assistance. W. Van den Ende is grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants.     

Unexpected presence of fructan 6-exohydrolases (6-FEHs) in non-fructan plants: characterization, cloning, mass mapping and functional analysis of a novel "cell-wall invertase-like" specific 6-FEH from sugar beet (Beta vulgaris L.)

About 15% of flowering plant species synthesize fructans. Fructans serve mainly as reserve carbohydrates and are subject to breakdown by plant fructan exohydrolases (FEHs), among which 1-FEHs (inulinases) and 6-FEHs (levanases) can be differentiated. This paper describes the unexpected finding that 6-FEHs also occur in plants that do not synthesize fructans. The purification, characterization, cloning and functional analysis of sugar beet (Beta vulgaris L.) 6-FEH are described. Enzyme activity measurements during sugar beet development suggest a constitutive expression of the gene in sugar beet roots. Classical enzyme purification followed by in-gel trypsin digestion and mass spectrometry (quadruple-time-of-flight mass spectrometry (Q-TOF) MS) led to peptide sequence information used in subsequent RT-PCR based cloning. Levan-type fructans (beta-2,6) are the best substrates for the enzyme, while inulin-type fructans (beta-2,1) and sucrose are poorly or not degraded. Sugar beet 6-FEH is more related to cell wall invertases than to vacuolar invertases and has a low iso-electric point (pI), clearly different from typical high pI cell wall invertases. Poor sequence homology to bacterial or fungal FEHs makes an endophytic origin highly unlikely. The functionality of the 6-FEH cDNA was further demonstrated by heterologous expression in Pichia pastoris. As fructans are absent in sugar beet, the role of 6-FEH in planta is not obvious. Like chitinases and beta-glucanases hydrolysing cell-surface components of fungal plant pathogens, a straightforward working hypothesis for further research might be that plant 6-FEHs participate in hydrolysis (or prevent the formation) of levan-containing slime surrounding endophytic or phytopathogenic bacteria.