Presence of Inulin-Type Fructo-Oligosaccharides and Shift from Raffinose Family Oligosaccharide to Fructan Metabolism in Leaves of Boxtree (Buxus sempervirens)

from raffinose family oligosaccharide to fructan metabolism in leaves of boxtree (Buxus sempervirens) Wim Van den Ende1,* Marlies Coopman1, Rudy Vergauwen1, André Van Laere11 KU Leuven, Laboratory of Molecular Plant Biology, Institute of Botany and Microbiology, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium* Correspondence: Wim Van den Ende, Laboratory of Molecular Plant Biology,Institute of Botany and Microbiology, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium tel +32 16321952; fax +32 16321967;[email protected]: inulin, oligosaccharides, stress, RFO, fructanAbstractFructans are known to occur in 15% of flowering plants and their accumulation is often associated with stress responses. Typically, particular fructan types occur within particular plant families. The family of the Buxaceae, harbouring Pachysandra terminalis, an accumulator of graminan- and levan-type fructans, also harbours boxtree (Buxus sempervirens), a cold and drought tolerant species. Surprisingly, boxtree leaves do not accumulate the expected graminan- and levan-type fructans but small inulin fructo-oligosaccharides (FOS: 1-kestotriose and nystose) and raffinose family oligosaccharides (RFO: raffinose and stachyose) instead. The seasonal variation in concentrations of glucose, fructose, sucrose, FOS and RFO were followed. Raffinose and stachyose peaked during the winter months, while FOS peaked at a very narrow time-interval in spring, immediately preceded by a prominent sucrose accumulation. Sucrose may function as a reserve carbohydrate in winter and early spring leaves. The switch from RFO to fructan metabolism in spring strongly suggests that fructan and RFO fulfil distinct roles in boxtree leaves. RFO may play a key role in the cold acclimation of winter leaves while temporal fructan biosynthesis in spring might increase sink strength to sustain the formation of new shoots

Presence of inulin-type fructo-oligosaccharides and shift from raffinose family oligosaccharide to fructan metabolism in leaves of boxtree (Buxus sempervirens)

Fructans are known to occur in 15% of flowering plants and their accumulation is often associated with stress responses. Typically, particular fructan types occur within particular plant families. The family of the Buxaceae, harboring Pachysandra terminalis, an accumulator of graminan- and levan-type fructans, also harbors boxtree (Buxus sempervirens), a cold and drought tolerant species. Surprisingly, boxtree leaves do not accumulate the expected graminan- and levan-type fructans, but small inulin fructo-oligosaccharides (FOS: 1-kestotriose and nystose) and raffinose family oligosaccharides (RFOs: raffinose and stachyose) instead. The seasonal variation in concentrations of glucose, fructose, sucrose, FOS and RFOs were followed. Raffinose and stachyose peaked during the winter months, while FOS peaked at a very narrow time-interval in spring, immediately preceded by a prominent sucrose accumulation. Sucrose may function as a reserve carbohydrate in winter and early spring leaves. The switch from RFO to fructan metabolism in spring strongly suggests that fructans and RFOs fulfill distinct roles in boxtree leaves. RFOs may play a key role in the cold acclimation of winter leaves while temporal fructan biosynthesis in spring might increase sink strength to sustain the formation of new shoots.status: publishe

Towards understanding vacuolar antioxidant mechanisms: a role for fructans?

Recent in vitro, in vivo and theoretical experiments strongly suggest that sugar-(like) molecules counteract oxidative stress by acting as genuine ROS scavengers. A concept was proposed to include the vacuole as a part of the cellular antioxidant network. According to this view sugars and sugar-like vacuolar compounds work in concert with vacuolar phenolic compounds and the “classic” cytosolic antioxidant mechanisms. Among the biologically relevant ROS (H2O2, O2•- and •OH), hydroxyl radicals are the most reactive and dangerous species since there are no enzymatic systems known to neutralize them in any living beings. Therefore, it is important to study in more detail the radical reactions between •OH and different biomolecules, including sugars. Here, we used Fenton reactions to compare the •OH scavenging capacities of a range of natural vacuolar compounds to establish relationships between antioxidant capacity and chemical structure and to unravel the mechanisms of •OH-carbohydrate reactions. The in vitro work on the •OH scavenging capacity of sugars and phenolic compounds revealed a correlation between structure and •OH scavenging capacity. The number and position of C=C type of linkages in phenolic compounds greatly influences antioxidant properties. Importantly, the splitting of disaccharides and oligosaccharides emerged as a predominant outcome of the •OH-carbohydrate interaction. Moreover, non-enzymatic synthesis of new fructan oligosaccharides was found starting from 1-kestotriose. Based on these and previous findings, a working model is proposed describing the putative radical reactions involving fructans and secondary metabolites at the inner side of the tonoplast and in the vacuolar lumen.status: publishe

Manninotriose is a major carbohydrate in red deadnettle (Lamium purpureum, Lamiaceae)

Background and aims The need is high to search for natural compounds with superior prebiotic, antioxidant and immunostimulatory properties for use in (food) applications. Raffinose Family Oligosaccharides (RFOs) show such properties. Moreover, they contribute to stress tolerance in plants acting as putative membrane stabilizers, antioxidants and signalling agents. Methods A large scale soluble carbohydrate screening was performed within the plant kingdom. An unknown compound accumulated to a high extent in early spring red deadnettle (Lamium purpureum) but not in other RFO plants. The compound was purified and its structure was unravelled with NMR. Organs and organ parts of red deadnettle were carefully dissected and analysed on soluble sugars. Phloem sap content was analysed by a common EDTA-based method. Key results Early spring red deadnettle stems and roots accumulate high concentrations of the reducing trisaccharide manninotriose (Galα1,6Galα1,6Glc), a derivative of the non-reducing RFO stachyose (Galα1,6Galα1,6Glcα1,2βFru). Detailed soluble carbohydrate analyses on dissected stem and leaf sections together with phloem sap analyses strongly suggest that stachyose is the main transport compound, but extensive hydrolysis of stachyose to manninotriose seems to occur along the transport path. Based on the specificities of the observed carbohydrate dynamics, the putative physiological roles of manninotriose in red deadnettle are discussed. Conclusions Here it is demonstrated for the first time that manninotriose is a novel and important player in the RFO metabolism of red dead deadnettle. It is proposed that manninotriose represents a temporal storage carbohydrate in early spring deadnettle, at the same time perhaps functioning as a membrane protector and/or as an antioxidant in the vicinity of membranes as recently suggested for other RFOs and fructans. This novel finding urges further research on this peculiar carbohydrate on a broader array of RFO accumulators.status: publishe

Building a fructan LC–MS2 library and its application to reveal the fine structure of cereal grain fructans

A liquid chromatography-mass spectrometry (LC–MS) library is presented containing the relative retention times of 28 fructan oligomers and MS2 spectra of 18 of them. It includes the main representativesof all fructan classes occurring in nature and with a degree of polymerization between three and five. This library enables a rapid and unambiguous detection of these 18 fructan structures in any type of sample without the need for fructan purification or the synthesis of fructan standards. Its wide applicability is demonstrated by the analysis of fructans in a set of cereal flour samples. Marked differences were observed in the types of fructans present in oat, barley, rye, spelt and wheat flour. A putative link between the accumulation of certain fructan types and cereal phylogeny is described.status: publishe

A simple and accurate method for determining wheat grain fructan content and average degree of polymerization

An improved method for the measurement of fructans in wheat grains is presented. A mild acid treatment is used for fructan hydrolysis, followed by analysis of the released glucose and fructose with high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Not only the amount of fructose set free from fructans but also the released glucose can be quantified accurately, allowing determination of the average degree of polymerization of fructans (DP(av)). Application of the mild acid treatment to different grain samples demonstrated that a correction should be made for the presence of sucrose and raffinose, but not for stachyose or higher raffinose oligosaccharides. The fructan content and DP(av) of spelt flour, wheat flour, and whole wheat flour were 0.6%, 1.2%, and 1.8% of the total weight and 4, 5, and 6, respectively. Validation experiments demonstrate that the proposed quantification method is accurate and repeatable and that also the DP(av) determination is precise.status: publishe