The maize Sh2r6hs ADP-glucose pyrophosphorylase (AGP) large subunit confers enhanced AGP properties in transgenic wheat ( Triticum aestivum)

Abstract

The rate of cereal seed starch biosynthesis is determined largely by the activity of starch biosynthetic enzymes within the endosperm. The heterotetrameric allosteric enzyme ADP-glucose pyrophosphorylase (AGP) controls the first committed step in starch synthesis, and is inhibited by phosphate (P i) and activated by 3-PGA. An altered AGP large subunit ( Sh2r6hs) from maize containing two alterations relative to the wild-type maize Sh2 sequence was introduced into wheat. The rev6 alteration confers reduced P i inhibition and decreased heat stability in maize and the hs alteration has been shown to increase AGP subunit interactions and enzyme stability in Escherichia coli. We have previously shown that low-level expression of Sh2r6hs under control of the maize Sh2 promoter increases yield in wheat and rice. Our objective here was to examine how Sh2r6hs affects AGP activity and yield in wheat and whether further yield increases were possible with greater Sh2r6hs expression. We therefore developed an additional population of transgenic wheat expressing SH2R6HS under control of the endosperm specific wheat Glutenin ( Glu) promoter. Yield increases in high-expressing Glu promoter Sh2r6hs lines were comparable to those with the Sh2 promoter despite the fact that the Glu promoter confers ∼20 times higher levels of Sh2r6hs expression than the Sh2 promoter and five times more SH2R6HS accumulation versus native wheat large AGP subunits. However, the high expression allowed endosperm AGP kinetic analysis. The I 0.5 of wheat AGP was increased from 2.0 to 4.1 mM P i by SH2R6HS expression and the A 0.5 was decreased from 0.82 to 0.31 mM 3-PGA. Two properties of Sh2r6hs AGP were identified that indicate greater subunit stability: wheat AGP small subunit levels were higher without variation in small subunit transcript levels, and in vitro activity was twice as great as untransformed AGP activity after heat treatment. Both r6 and hs alterations act independently and synergistically to modify AGP allosteric-regulation, increase AGP subunit stability, and increase yield in plants expressing Sh2r6hs.