Molecular Characterization of the Brittle-2 Gene Effect on Maize Endosperm ADPglucose Pyrophosphorylase Subunits

Activity of the enzyme ADPglucose pyrophosphorylase is known to be reduced in maize (Zea mays L.) endosperm mutants at two independent loci, Shrunken-2 (Sh(2)) and Brittle-2 (Bt(2)). Spinach leaf ADPglucose pyrophosphorylase has previously been shown to comprise two subunits of 51 and 54 kilodaltons. Anti-bodies raised to each of the two subunits of spinach leaf ADPglucose pyrophosphorylase were found to cross-react to different bands on Western blots prepared from polyacrylamide gel electrophoresis separated wild-type maize endosperm proteins. The anti-spinach leaf 51 kilodalton subunit antibody cross-reacted with a 55 kilodalton maize endosperm protein and the anti-spinach leaf 54 kilodalton subunit antibody cross-reacted with a 60 kilodalton maize endosperm protein. These immunological reactions were observed in maize endosperm extracts and with a highly purified preparation of maize endosperm ADPglucose pyrophosphorylase. Mutant bt(2) endosperm lacked the 55 kilodalton subunit while mutant sh(2) endosperm lacked the 60 kilodalton subunit on Western blots. These results suggest that the maize endosperm ADPglucose pyrophosphorylase is made up of two immunologically dissimilar subunits and that the bt(2) and sh(2) mutations cause reduction in ADPglucose pyrophosphorylase activity through the lack of one of these two subunits. An ADPglucose pyrophosphorylase cDNA clone antigenically selected from a rice seed cDNA expression library was found to hybridize strongly with a cDNA corresponding to a maize endosperm transcript which is absent in a W64A bt(2) mutant. Thus, the bt(2) mutant causes the absence not only of the small subunit but of the corresponding transcript. Bt(2) is implicated as the structural gene for the small (54 kilodalton) subunit of maize endosperm ADPglucose pyrophosphorylase.