title: Cloning, characterization and functional analysis of novel 6-kestose exohydrolases (6-KEHs) from wheat (Triticum aestivum).
abstract: 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.
authors.affiliation: K.U. Leuven, Institute of Botany and Microbiology, Laboratory of Molecular Plant Physiology, Kasteelpark Arenberg 31, B-3001 Heverlee, Belgium. vandenende@bio.kuleuven.ac.be
authors.forename: Wim
authors.initials: W
authors.lastname: Van den Ende
authors.forename: Midori
authors.initials: M
authors.lastname: Yoshida
authors.forename: Stefan
authors.initials: S
authors.lastname: Clerens
authors.forename: Rudy
authors.initials: R
authors.lastname: Vergauwen
authors.forename: Akira
authors.initials: A
authors.lastname: Kawakami
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes; D008969:Molecular Sequence Data; D010802:Phylogeny; D036226:Seedlings / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology* / Q000254:growth & development
chemical_list: D007527:Isoenzymes; D006026:Glycoside Hydrolases