Mutation at the shrunken-2 (Sh2) locus of maize, a gene described more than 40 years ago, greatly reduces starch levels in the endosperm through its effect on the starch synthetic enzyme ADP-glucose pyrophosphorylase, an enzyme thought to be regulatory in this biosynthetic pathway. Although our previous work has suggested that Sh2 is a structural gene for this enzyme, we have also reported data compatible with Sh2 acting post-transcriptionally.In this study, we took advantage of a transposable element-induced Sh2 allele, its progenitor, and revertants to identify a clone for this locus. Although the cloning and identification were done independently of any knowledge concerning the product of this gene, examination of the deduced amino acid sequence revealed much similarity to known ADP-glucose pyrophosphorylase subunits of plants and bacteria, including regions involved in substrate binding and activator binding. Little sequence similarity, however, was found at the DNA level. These obsenrations provide direct evidence that Sh2 encodes a subunit for endosperm ADP-glucose pyrophosphorylase. Analysis of several phenotypically wild-type alleles arising from a mutable sh2-Ds allele revealed one unexpected case in which DNA sequences of Sh2 were rearranged in comparison with the progenitor Sh2. In contrast to wild type, the Dsinduced sh2 allele conditions at least two transcripts in the endosperm.

Activity of the enzyme ADPglucose pyrophosphorylase is known to be reduced in maize (Zea mays L.) endosperm mutants at two independent loci, Shrunken-2 (Sh2) and Brittle-2 (Bt2). Spinach leaf ADPglucose pyrophosphorylase has previously been shown to comprise two subunits of 51 and 54 kilodaltons. Antibodies 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 antispinach 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 bt2 endosperm lacked the 55 kilodalton subunit while mutant sh2 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 bt2 and sh2 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 bt2 mutant. Thus, the bt2 mutant causes the absence not only of the small subunit but of the corresponding transcript. Bt2 is implicated as the structural gene for the small (54 kilodalton) subunit of maize endosperm ADPglucose pyrophosphorylase.ADPglucose pyrophosphorylase has been implicated as a key regulatory enzyme for starch biosynthesis in both leafand storage tissues (14). Mutation at the independent loci Shrunken-2 (Sh2) and Brittle-2 (Bt) results in dramatic reduction of maize endosperm ADPglucose pyrophosphorylase activity (7,17) as well as approximately 25% of normal starch content (4,6,17). However, the mechanism by which these mutants cause reduction of enzyme activity had not previously been established.

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Equimolar reaction of the title ligand (II) with the (norbornadiene)tetracarbonyls (I) results in the formation of the monosubstitution products (III).