Cloning, characterization and functional analysis of novel 6‐kestose exohydrolases (6‐KEHs) from wheat (Triticum aestivum)

Abstract

Summary• Cereals accumulate graminan-type fructans which are subject to stress-related degradation by fructan 1-exohydrolases (1-FEHs) and fructan 6-exohydrolases (6-FEHs). To find new FEH genes related to freezing tolerance, a cold-hardened wheat crown cDNA library was screened.• Here we report the cloning, purification and characterization of two novel 6-kestosidase (6-KEH) isoenzymes from wheat crowns ( Triticum aestivum ). Functional characterization in Pichia pastoris confirmed the extreme substrate selectivity for the fructan trisaccharide 6-kestose.• Northern blotting showed that 6-KEH transcripts were constantly detected at the same level from autumn to winter in crown but not in leaf tissues. Apoplastic fluid isolations and activity measurements strongly suggest that 6-KEH is localized in the apoplast.• It is proposed that 6-KEHs, together with other FEHs, might be involved in the breakdown of apoplastic fructans which may fulfil a role as membrane protectors under stress. Alternatively, a role in signalling processes, or in the degradation of exogenous 6-kestose from bacterial origin, cannot be excluded.

Molecular Dissection of Variation in Carbohydrate Metabolism Related to Water-Soluble Carbohydrate Accumulation in Stems of Wheat

“…Fructan is hydrolyzed to Fru and Suc by fructan 1-exohydrolase (1-FEH) and/or fructan 6-exohydrolase (6-FEH; Van den Ende et al, 2005;Van Riet et al, 2006). The transcript levels of TaFEH genes did not appear to be associated with genotypic variation in the stem WSCs at anthesis.…”

Research on Fructan in Wheat and Temperate Forage Grasses in Japan

“…Kawakami and Yoshida 8,9 cloned 4 cDNAs that cover all kinds of fructan synthetic enzymes in wheat. Several clones of wheat fructan exohydrolases, 1-FEH 22,25 , 6-FEH (6-KEH) 23,24 , and 6&1-FEH 10 , have been isolated and functionally analyzed. Plant fructosyltransferases and fructan exohydrolases show high homology to vacuolar and cell-wall invertases, respectively (Fig.…”

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

“…Enzymes that preferentially degrade fructan trisaccharides were recently discovered in fructan plants. Specific 6-kestosidase (6-KEH) enzymes have been purified and cloned from wheat (Triticum aestivum; Van den Ende et al, 2005) and 1-KEH activities were reported in onion (Allium cepa; Benkeblia et al, 2005). Strikingly, the D239A mutant showed superior affinity (K m 600 mM) for 1-kestose compared to the naturally occurring FEHs from chicory (1-FEH IIa: 58 mM; De Roover et al, 1999) and wheat (1-FEH w1 and w2: 7 mM; Van den Ende et al, 2003a).…”