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 endospermspecific B1-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 B1-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 ␣-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.

Protein Phosphorylation in Amyloplasts Regulates Starch Branching Enzyme Activity and Protein–Protein Interactions

“…Redox control of AGPase in Solanum tuberosum (potato) tubers has been proposed recently (Fu et al, 1998), and further work demonstrated that this phenomenon is relatively widespread and includes photosynthetic and nonphotosynthetic tissues from other species (Tiessen et al, 2002;Hendriks et al, 2003). There also is evidence for redox regulation of pullulanase-type DBEs in several tissues, including Spinacia oleracea (spinach) leaves and the endosperms of Hordeum vulgare (barley) and maize (Beatty et al, 1999;Cho et al, 1999;Schindler et al, 2001).…”

An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

“…Finally, the protein disulfide reductase thioredoxin has been proposed to regulate the amylolytic system in barley seeds by reduction of disulfide bonds in enzymes and inhibitors (Cho et al 1999). Barley contains two thioredoxin isoforms as well as two isoforms of an NADPH-dependent thioredoxin reductase that reduce the disulfide formed in the thioredoxin active site motif CXXC after it has reduced a target protein disulfide bond (Maeda et al 2003;Shahpiri et al 2008).…”

Thioredoxin in Barley: Could It Have a Role in Releasing Limit Dextrinase in Brewery Mashes?

“…After centrifugation for 15 min at 14,000 rpm at 4°C on an Eppendorf 5415C centrifuge (Hamburg, Germany), the supernatant was stored on ice before assaying for protein 2 (by the Coomassie Blue dyebinding assay with gamma globulin as standard) and thioredoxin 4 . The latter involves separation of proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with a positive thioredoxin control from transgenic barley followed by Western blotting.…”

“…It was shown that over-expression of thioredoxin in barley greatly enhanced the level of LD expressed in the barley 4 . It was concluded that stimulation could not be traced to the reduction and inactivation of the inhibitor and it was suggested that the mechanism was either an increase in the de novo synthesis of the enzyme or by a promotion of the release of the enzyme from binding.…”