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 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.

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.