ADPGlucose pyrophosphorylase (E.C.2.7.7.9) catalyzes the conversion G-1-P + ATP 4 <--> ADPGlucose +PPi, which is the regulatory step in the formation of starch in plants. Activation by photosynthetic intermediates (3-PGA, fructose-1,6 diphosphate, fructose-6 phosphate) and inhibition by orthophosphate and ADP serve to control ADPGlucose pyrophosphorylase activity and, thus, the rate of starch accumulation in endosperm and leaf tissue.
The starch deficient mutants sh2 and bt2 represent two structural genes which code for ADPGlucose pyrophosphorylase in maize endosperm in maize endosperm (Hannah and Nelson, Plant Physiol. 53:297-302, 1975). The enzyme exists in two forms, A and B, in endosperm, which represent different aggregation states rather than different molecular species. The B form is a dimer composed of one subunit encoded by each structural gene while the A form is a tetramer composed of two subunits from each structural gene (Fuchs and Smith, Plant Physiol., submitted).
Comparisons of endosperm ADPGlucose pyrophosphorylase activities were made at 10, 13, 16 and 19 days post pollination among nine endosperm mutants and a commercial hybrid. The mutants assayed were ae, bt2, fl, o, o2, sh2, su, and wx, while TX40 was used as the normal endosperm source. Ears were frozen when harvested and stored at -10 to -15 C until needed. The procedures for measuring ADPGlucose synthesis and isolation of ADPGlucose followed the method of Dickinson and Preiss (Arch. Biochem. Biophys. 130:119-128, 1969) as modified by Hannah and Nelson (Plant Physiol. 53:297-302, 1975) and Fuchs and Smith (Plant Physiol., submitted). One unit of enzyme activity is equal to 1 µmole ADPGlucose formed in 30 minutes. Protein was estimated by the method of Lowry et al. (J. Biol. Chem. 193:263-275, 1951) with BSA as the standard.
The bt2 and sh2 mutants, which were known to affect ADPGlucose pyrophosphorylase, were the only mutants whose activity was significantly lower than normal (TX40) at all stages of development. In general, activity increased with age for the other genotypes, but appreciable variation in activity was observed over time. Opaque and sh had significantly higher activity than TX40, while su and wx were of interest since activity was essentially normal at 13 days post pollination but did not increase during the later developmental stages. Caution must be used in interpreting these data, however, as an inhibitor of ADPGlucose pyrophosphorylase is present in the crude extract (Fuchs and Smith, Plant Physiol., submitted). Since these assays were run on crude extracts, it is not possible to conclude whether these observations represent differences in enzyme or inhibitor levels at this time.
James D. Smith and Roy L. Fuchs
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