The Sh locus on chromosome 9 has been shown to be the structural gene for the major form of sucrose synthetase in the developing endosperm of maize (Chourey and Nelson, 1976, Biochem. Genet. 14:1041). This form of the enzyme was found missing in the shrunken (sh/sh) endosperm. The residual sucrose synthetase activity in the mutant endosperm was thought to be due to another locus elsewhere in the genome. According to this, the normal endosperm should have both forms of the enzyme unless some complex gene interaction/regulation is taking place. However, to date, it has not been possible to conclusively demonstrate the presence of two forms. The observations described in this report concern the characterization of sucrose synthetase activity in normal and shrunken endosperms and normal embryos.
The enzyme sucrose synthetase is known to catalyze sucrose synthesis as well as sucrose cleavage. Both these reactions can be readily assayed. Experiments were done to determine the pH optimums for the two reactions. Sucrose synthesis and sucrose cleavage reactions showed two distinctly different pH optimums; but no differences were found for the two genotypes as well as the embryos (Table 1). It was noticed during these studies that the HEPES buffer which was used for all the experiments described in the previous report (Biochem. Genet. 14:1041) was inhibitory in action as compared to the tris-chloride buffer (same pH). In addition, the sucrose cleavage reaction was previously underestimated in view of the low pH optimum described here. This necessitated a re-examination of the enzyme activity in various shrunken mutants (Table 2) using glycine-NaOH buffer for sucrose synthesis and MES buffer for sucrose cleavage.
All the sh mutants described in Table 2 are of independent origin and have a residual activity of less than 5% as compared to the normal endosperm. The embryos of both the genotypes have approximately 15% enzyme activity (as measured in either direction) of the normal endosperm. Various properties of the enzyme in the endosperm and embryo are listed in Table 1. The sh/sh endosperm enzyme is distinguishable from the Sh/Sh endosperm by only two criteria: 1) a very slight difference in electrophoretic mobility and 2) that the former shows a lesser amount of sucrose synthetase protein by electrophoretic as well as immunochemical criteria.
Immunochemical cross-reactivity was judged in the past by Ouchterlony double diffusion tests. No evidence of the presence of any protein that will cross-react with the Sh protein was seen in the sh endosperm and the embryos (of either genotype). In the present studies we have assayed the same property by immunoinactivation of the enzyme activity after the treatment of an enzyme with an antiserum. A complete inactivation of the enzyme activity was observed when crude preparations of Sh or sh endosperm or embryos were treated with an antiserum raised against the partially purified Sh protein. The antiserum raised against the partially purified sucrose synthetase from sh/sh endosperm, when treated similarly, also led to the complete inactivation of enzyme activity. Reasons for the discrepancy between the two immunological tests are not known. The developmental profile was monitored starting from 4 days through 48 days after pollination and no qualitative differences between the two genotypes were observed.
Prem S. Chourey and Oliver E. Nelson
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