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Media type:
E-Article
Title:
Overexpression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain
Contributor:
Cho, Myeong-Je;
Wong, Joshua H.;
Marx, Corina;
Jiang, Wen;
Lemaux, Peggy G.;
Buchanan, Bob B.
imprint:
Proceedings of the National Academy of Sciences, 1999
Published in:Proceedings of the National Academy of Sciences
Language:
English
DOI:
10.1073/pnas.96.25.14641
ISSN:
1091-6490;
0027-8424
Origination:
Footnote:
Description:
<jats:p>
Biochemically active wheat thioredoxin
<jats:italic>h</jats:italic>
has been overexpressed in the endosperm of transgenic barley grain. Two DNA constructs containing the wheat thioredoxin
<jats:italic>h</jats:italic>
gene (
<jats:italic>wtrxh</jats:italic>
) were used for transformation; each contained
<jats:italic>wtrxh</jats:italic>
fused to an endosperm-specific B
<jats:sub>1</jats:sub>
-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
<jats:italic>bar</jats:italic>
herbicide resistance gene on a cotransformed plasmid; all were positive for this gene. The presence of
<jats:italic>wtrxh</jats:italic>
was confirmed in 20 lines by PCR analysis, and the identity and level of expression of wheat thioredoxin
<jats:italic>h</jats:italic>
was assessed by immunoblots. Although levels varied among the different transgenic events, wheat thioredoxin
<jats:italic>h</jats:italic>
was consistently highly expressed (up to 30-fold) in the transgenic grain. Transgenic lines transformed with the B
<jats:sub>1</jats:sub>
-hordein promoter with a signal peptide sequence produced a higher level of wheat thioredoxin
<jats:italic>h</jats:italic>
on average than those without a signal sequence. The overexpression of thioredoxin
<jats:italic>h</jats:italic>
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 α-1,6 linkages in starch. These results raise the question of how thioredoxin
<jats:italic>h</jats:italic>
enhances the activity of pullulanase because it was found that the inhibitor had become inactive before the enzyme showed appreciable activity.
</jats:p>