Towards understanding vacuolar antioxidant mechanisms: a role for fructans?

Abstract

Recent in vitro, in vivo, and theoretical experiments strongly suggest that sugar-(like) molecules counteract oxidative stress by acting as genuine reactive oxygen species (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 (H(2)O(2), O(2)·(-), 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, Fenton reactions were used 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 the C=C type of linkages in phenolic compounds greatly influence 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.

Fig. 1.

HPAEC-PAD chromatograms showing the products derived from in vitro Fenton reagent–carboydrate reactions. (A) The tested carbohydrates before the onset of the Fenton reactions are as follows: 1, trehalose (Tre); 2, sucrose (Suc); 3, maltose (Mal), maltotriose (Maltri); 4, raffinose (Raf); 5, 1-kestotriose (1-K); 1,1-nystose (N). (B) The carbohydrate mixtures derived after the Fenton reactions: 1, trehalose; 2, sucrose; 3, maltose; 4, raffinose; 5, 1-kestotriose; 6, a reference containing trehalose (Tre), glucose (Glc), fructose (Fru), melibiose (Mel), and sucrose (Suc).

Fig. 2.

Some possible reactions between sucrose and the ·OH radical leading to the scission of the glycosidic bond. The ·OH radical abstracts a hydrogen atom (H) from sucrose yielding a sugar radical and a free hexose. The abstraction may occur at the glucose or fructose moiety, respectively. Three possible reactions are presented: (i) The radical attack occurs on the glucose moiety next to the glycosidic bond (reaction 2). In this case a direct scission of the bond occurs and a glucose radical and fructose are released. (ii) The H is abstracted from the fructose or glucose moiety but not in the neighbourhood of the glycosidic linkage (reactions 1 and 3). Probably, first a ring opening takes place in the corresponding moiety followed by the scission of the glycosidic bond. In all cases, a free hexose and a hexose radical are formed.

Fig. 3.

Effect of 1-FEH and 6-FEH enzyme activity on the products generated during a Fenton reaction with 1-kestotriose. (1) A reference containing glucose (Glc), fructose (Fru), sucrose (Suc), 1-kestotriose (1-K), and 1,1-nystose (N). (2) Products generated by a Fenton reaction with 1-kestotriose (F₂ and F₃). (3) Products as depicted under 2 were further incubated with chicory 1-FEH IIa for 24h. (4) Products as depicted under 2 were further incubated with sugar beet 6-FEH for 24h. (5) Products as depicted under 2 were further incubated with chicory 1-FEH IIa and sugar beet 6-FEH for 24h.

Fig. 4.

Purification of F₂ and F₃ from the 1-kestotriose Fenton reaction mixture. The molecular weight (MW) detected by mass spectroscopy is indicated on the HPAEC-PAD profiles. (1) A reference containing glucose (Glc), fructose (Fru), sucrose (Suc), 1-kestotriose (1-K), and, 1,1-nystose (N). (2) A forced chicory root extract. (3) The HPAEC-PAD profile of purified F₂; the MW obtained by mass spectrometry and corresponding to the disaccharide-Na adduct is shown. (4) Co-injection of purified F₂ with a forced chicory root extract. (5) The HPAEC-PAD profile of purified F₃: the MW obtained by mass spectrometry analysis corresponding to the trisaccharide-Na adduct is presented. (6) Co-injection of purified F₃ with a forced chicory root extract.

Fig. 5.

Schematic presentation of non-enzymatic oligosaccharide synthesis from 1-kestotriose in a Fenton reaction. The ·OH radical abstracts a hydrogen (H) atom from 1-kestotriose, leading to a formation of a 1-kestotriose radical which (partially) fragments into lower DP sugars and sugar radicals. The latter might recombine with each other forming novel, higher DP oligosaccharides. Thus, both breakdown and synthesis of new oligosaccharides can occur in radical reactions involving ·OH and 1-kestotriose. Free radical molecules are indicated by stars.

Fig. 6.

HPAEC-PAD sugar profiles of saps derived from chicory leaves. (A) Control leaves. (B) Excised leaves incubated for 2 d in water. (C) Excised leaves incubated for 2 d in 250mM Suc. Glc, glucose; Fru, fructose; Suc, sucrose; 1K, 1-kestotriose; N, 1,1-nystose; 5, 1,1,1-kestopentaose.

Fig. 7.

Model for the role of fructans in the vicinity of tonoplastic membranes and integration in the cellular antioxidant network. Two distinct areas can be distinguished in vacuoles: the near tonoplast inner space and the central vacuolar lumen. Fructans and phenolics are both vacuolar compounds. Fructans strongly interact with membranes and thus a higher concentration can be expected in the near tonoplast environment, while phenolic compounds might predominate in the vacuolar lumen. Under stress, excess cytosolic H₂O₂ might pass through the tonoplast (either by diffusion or assisted by aquaporins) and enter the vacuole. Additionally, superoxide radicals (O₂·^–) may be produced by tonoplast-resident NADPH oxidases and transformed to vacuolar H₂O₂ by superoxide dismutase (SOD). H₂O₂ is a substrate of type III vacuolar peroxidases (PRXs) associated with the tonoplast. PRXs may produce ·OH through the hydroxylic cycle. Inserting deep into the tonoplastic membrane, fructans are ideally positioned to react with this radical, resulting in the formation of new carbohydrate radicals. As deduced from our in vitro experiments, these radicals may undergo scission, splitting up into smaller radical and non-radical components that tend to diffuse away from the tonoplastic membrane into the central vacuolar lumen. Here, sugar radicals might be recycled to sugars and/or radical recombination reactions may occur, resulting in the formation of sugar–phenol compounds, higher DP neutral carbohydrates, or phenolics. Furthermore, sugar recycling might occur at the expense of secondary metabolites (e.g. phenolic compounds) that need subsequent recycling on their own with ascorbate (AsA) and/or glutathione (GSH) that seem to be present at least in some vacuoles. However, further research is needed to demonstrate the presence of AsA and GSH transporters in the tonoplast.