Overexpression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain

Biochemically active wheat thioredoxin h has been overexpressed in the endo sperm of transgenic barley grain. Two DNA constructs containing the wheat t hioredoxin h gene (wtrxh) were used for transformation; each contained wtrx h fused to an endosperm-specific B-1-hordein promoter either with or withou t a signal peptide sequence for targeting to the protein body. Twenty-two s table, independently transformed regenerable lines were obtained by selecti ng with the herbicide bialaphos to test for the presence of the bar herbici de resistance gene on a cotransformed plasmid; all were positive for this g ene. The presence of wtrxh was confirmed in 20 lines by PCR analysis, and t he 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 th e transgenic grain. Transgenic lines transformed with the B-1-hordein promo ter with a signal peptide sequence produced a higher level of wheat thiored oxin 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-fo ld 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 t he activity of pullulanase because it was found that the inhibitor had beco me inactive before the enzyme showed appreciable activity.