On the Roles of Wheat Endosperm ADP-Glucose Pyrophosphorylase Subunits

Abstract

The ADP-glucose pyrophosphorylase from wheat endosperm controls starch synthesis in seeds and has unique regulatory properties compared to others from this family. It comprises two types of subunits, but despite its importance little is known about their roles. Here, we synthesized de novo the wheat endosperm ADP-glucose pyrophosphorylase small (S) and large (L) subunit genes, heterologously expressed them in Escherichia coli, and kinetically characterized the recombinant proteins. To understand their distinct roles, we co-expressed them with well characterized subunits from the potato tuber enzyme to obtain hybrids with one S subunit from one source and an L subunit from the other. After kinetic analyses of these hybrids, we concluded that the unusual insensitivity to activation of the wheat endosperm enzyme is caused by a pre-activation of the L subunit. In addition, the heat stability and sensitivity to phosphate are given by the S subunit.

Phosphorylation of ADP-Glucose Pyrophosphorylase During Wheat Seeds Development

“…Also, in the analysis of the samples from the incubation of Tae S or Tae L subunits with the recombinant plant protein kinases using the approach of western-blots from SDS-PAGE with Phos-tag, the lower mobility was only observed for L polypeptide ( Supplemental Figure 4 ). These results strongly agree with the key (tissue-specific) regulatory role assigned to the L subunit of heterotetrameric ADP-Glc PPases ( Crevillén et al., 2003 ; Ballicora et al., 2004 ; Ventriglia et al., 2008 ; Ferrero et al., 2018 ; Figueroa et al., 2018 ), suggesting that its post-translational modification by phosphorylation would be relevant for starch synthesis in wheat seeds.…”

“…The genes coding for the small ( Tae S) and large ( Tae L) subunits of wheat endosperm ADP-Glc PPase were synthesized de novo from DNA sequence reported previously ( Ainsworth et al., 1993 and Ainsworth et al., 1995 ) and subcloned into pET28c as reported elsewhere ( Ferrero et al., 2018 ). This recombinant enzyme displayed similar properties to the ADP-Glc PPase purified from the wheat endosperm ( Gómez-Casati and Iglesias, 2002 ).…”

“…For in vitro phosphorylation of recombinant forms of the wheat endosperm ADP-Glc PPase [the heterotetrameric ( Tae SL), small subunit ( Tae S), or large subunit ( Tae L); see ( Ferrero et al., 2018 ), the respective purified enzyme (2 µg) was incubated under conditions established for the activity of different protein kinases from plants ( Piattoni et al., 2011 ; Rojas et al., 2018 )]. The three (I–III) specific phosphorylation conditions used for assay crude extracts from seeds or purified recombinant protein kinase were as follows.…”

“…The ADP-Glc PPase presents in green algae and higher plants is a heterotetramer (S 2 L 2 ), composed of small (S, 50-53 kDa) and large (L, 54-60 kDa) subunits ( Ballicora et al., 2003 ; Ballicora et al., 2004 ). Subunits S and L are homologous proteins, where the L polypeptide has emerged in different species via gene duplication followed by subfunctionalization ( Ballicora et al., 2005 ; Georgelis et al., 2008 ; Georgelis et al., 2009 ; Kuhn et al., 2009 ; Ferrero et al., 2018 ; Figueroa et al., 2018 ). In potato tuber, the S subunit retained the catalytic function, whereas the L subunit specialized in modulating the regulation of the former ( Ballicora et al., 2005 ).…”

“…In the barley and maize forms, 3PGA modifies the relative affinity for substrates ( Plaxton and Preiss, 1987 ; Kleczkowski et al., 1993 ). Also, in wheat ( Triticum aestivum) endosperm enzyme, neither 3PGA nor fructose-6P (Fru6P) modify the V max , but they increase the relative affinity for Glc1P by 2-fold ( Gómez-Casati and Iglesias, 2002 ; Ferrero et al., 2018 ). On the other hand, the 3PGA and Fru6P revert the inhibition by Pi ( Gómez-Casati and Iglesias, 2002 ).…”