title: Pp6-FEH1 encodes an enzyme for degradation of highly polymerized levan and is transcriptionally induced by defoliation in timothy (Phleum pratense L.).
abstract: The ability of grasses to regrow after defoliation by cutting or grazing is a vital factor in their survival and an important trait when they are used as forage crops. In temperate grass species accumulating fructans, defoliation induces the activity of a fructan exohydrolase (FEH) that degrades fructans to serve as a carbon source for regrowth. Here, a cDNA from timothy was cloned, named Pp6-FEH1, that showed similarity to wheat fructan 6-exohydrolase (6-FEH). The recombinant enzyme expressed in Pichia pastoris completely degraded fructans that were composed mainly of β(2,6)-linked and linear fructans (levan) with a high degree of polymerization (DP) in the crown tissues of timothy. The substrate specificity of Pp6-FEH1 differed from previously characterized enzymes with 6-FEH activity in fructan-accumulating plants: (i) Pp6-FEH1 showed 6-FEH activity against levan (mean DP 20) that was 4-fold higher than against 6-kestotriose (DP 3), indicating that Pp6-FEH1 has a preference for β(2,6)-linked fructans with high DP; (ii) Pp6-FEH1 had significant activity against β(2,1)-linked fructans, but considerably less than against β(2,6)-linked fructans; (iii) Pp6-FEH1 had weak invertase activity, and its 6-FEH activity was inhibited slightly by sucrose. In the stubble of seedlings and in young haplocorms from adult timothy plants, transcripts of Pp6-FEH1 were significantly increased within 3 h of defoliation, followed by an increase in 6-FEH activity and in the degradation of fructans. These results suggest that Pp6-FEH1 plays a role in the degradation of fructans and the mobilization of carbon sources for regrowth after defoliation in timothy.
authors.affiliation: National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira, Sapporo 062-8555, Japan. tamuken@affrc.go.jp
authors.forename: Ken-ihi
authors.initials: KI
authors.lastname: Tamura
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Sanada
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Tase
authors.forename: Toshinori
authors.initials: T
authors.lastname: Komatsu
authors.forename: Midori
authors.initials: M
authors.lastname: Yoshida
mesh_terms: D005630:Fructans / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant / Q000235:genetics / Q000502:physiology; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D031784:Phleum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D005630:Fructans; D010940:Plant Proteins; C072599:levan; D006026:Glycoside Hydrolases