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.)

Abstract

SummaryAbout 15% of¯owering 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 ®nding that 6-FEHs also occur in plants that do not synthesize fructans. The puri®cation, 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 puri®ca-tion followed by in-gel trypsin digestion and mass spectrometry (quadruple-time-of-¯ight mass spectrometry (Q-TOF) MS) led to peptide sequence information used in subsequent RT-PCR based cloning. Levantype fructans (b-2,6) are the best substrates for the enzyme, while inulin-type fructans (b-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 bglucanases 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.

Unexpected Presence of Graminan- and Levan-Type Fructans in the Evergreen Frost-Hardy Eudicot Pachysandra terminalis (Buxaceae): Purification, Cloning, and Functional Analysis of a 6-SST/6-SFT Enzyme

“…8A). This finding is in line with previous observations that some vacuolar invertases show intrinsic FEH activities (Van den Ende et al, 2003a;De Coninck et al, 2005;Ji et al, 2007), suggesting that perhaps many vacuolar invertases have such characteristics. This new point of view sheds a different light on some former data and conclusions.…”

“…As a result of a major screening of .100 plant species belonging to different families in the plant kingdom, by means of high-performance anion-exchange chromatography and pulsed-amperometric detection (HPAEC-PAD), the rhizomes of P. terminalis were found to contain a complicated mixture of so far unidentified oligo-and polysaccharides in Figure 1. When a neutral, carbohydrate-containing fraction of P. terminalis rhizomes was subjected to mild acid and enzymatic hydrolysis with chicory (Cichorium intybus) 1-FEH IIa ( Van den Ende et al, 2001) and sugar beet (Beta vulgaris) 6-FEH (Van den Ende et al, 2003a), a massive production of Fru was observed (data not shown). A closer inspection of the oligosaccharides from P. terminalis on the HPAEC-PAD chromatograms revealed that it contained (1) a series of peaks corresponding to the graminan-type Figure 1.…”

Unraveling the Difference between Invertases and Fructan Exohydrolases: A Single Amino Acid (Asp-239) Substitution Transforms Arabidopsis Cell Wall Invertase1 into a Fructan 1-Exohydrolase

“…Without exception, all functionally characterized FEHs lack an Asp-239 homolog and are unable to break down Suc (Van den Ende et al, 2000, 2001, 2003a, 2003bDe Coninck et al, 2005;Van Riet et al, 2006). On the contrary, the presence of such an Asp-239 homolog seems to be a general feature of cell wall invertases.…”

Circadian and developmental regulation of vacuolar invertase expression in petioles of sugar beet plants

“…One of the cell wall invertase genes (CWI-1) was rapidly induced in wounded roots, whereas one of the vacuolar invertase genes (VI-1; Accession number AJ277457) showed a delayed induction, coincident with the increase in fructose and glucose in wounded roots. More recently, a cell wall invertase-like specific fructan 6-exohydrolase has been cloned and characterized from sugar beet (Van den Ende et al 2003). Since sugar beet plants do not synthesize fructans, the function of this novel enzyme is not yet clear.…”