title: Overexpression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain.
abstract: Biochemically active wheat thioredoxin h has been overexpressed in the endosperm of transgenic barley grain. Two DNA constructs containing the wheat thioredoxin h gene (wtrxh) were used for transformation; each contained wtrxh fused to an endosperm-specific B(1)-hordein promoter either with or without a signal peptide sequence for targeting to the protein body. Twenty-two stable, independently transformed regenerable lines were obtained by selecting with the herbicide bialaphos to test for the presence of the bar herbicide resistance gene on a cotransformed plasmid; all were positive for this gene. The presence of wtrxh was confirmed in 20 lines by PCR analysis, and the identity and level of expression of wheat thioredoxin h was assessed by immunoblots. Although levels varied among the different transgenic events, wheat thioredoxin h was consistently highly expressed (up to 30-fold) in the transgenic grain. Transgenic lines transformed with the B(1)-hordein promoter with a signal peptide sequence produced a higher level of wheat thioredoxin h on average than those without a signal sequence. The overexpression of thioredoxin h in the endosperm of germinated grain effected up to a 4-fold increase in the activity of the starch debranching enzyme, pullulanase (limit dextrinase), the enzyme that specifically cleaves alpha-1,6 linkages in starch. These results raise the question of how thioredoxin h enhances the activity of pullulanase because it was found that the inhibitor had become inactive before the enzyme showed appreciable activity.
authors.affiliation: Department of Plant Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Cho
authors.forename: J H
authors.initials: JH
authors.lastname: Wong
authors.forename: C
authors.initials: C
authors.lastname: Marx
authors.forename: W
authors.initials: W
authors.lastname: Jiang
authors.forename: P G
authors.initials: PG
authors.lastname: Lemaux
authors.forename: B B
authors.initials: BB
authors.lastname: Buchanan
mesh_terms: D006026:Glycoside Hydrolases / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology*; D030821:Plants, Genetically Modified; D054480:Thioredoxin h; D013879:Thioredoxins / Q000032:analysis*; D014170:Transformation, Genetic
chemical_list: D054480:Thioredoxin h; D013879:Thioredoxins; D006026:Glycoside Hydrolases; C100181:pullulanase