The brittle-1 (bt1) mutant was first described by Mangelsdorf in 1926. The gross phenotypic effect is very similar to bt2 and sh2 in producing a shrunken endosperm of brittle texture, and intermediate in translucency between normal and su1. The starch content was found to be markedly lower in bt1 seed than in normal at all stages of development. Large differences between bt1 and normal were also found for sucrose and reducing sugars during development. The specific biochemical lesion of the bt1 locus has not been known. We report here that bt1 endosperms are deficient in a starch granule-bound enzyme, phospho-oligosaccharide synthase, relative to nonmutant endosperms. Phospho-oligosaccharide synthase is an enzyme which converts Glc-1-P into a series of short chain length (G2 to G10) phospho-oligosaccharides and oligosaccharides (Pan and Nelson, ms. in preparation). Starch granules of developing kernels (22 days after self-pollination) of a normal inbred line (W64A) and mutant bt1 were isolated by washing 4 times with 50mM glycylglycine buffer, pH 8, and finally with cold acetone. The solubilization of phospho-oligosaccharide synthase from starch granules was based on the method of F.D. MacDonald and J. Preiss (Plant Physiol. 73: 175-178). The proteins solubilized from starch granules were then precipitated with ammonium sulfate (60%), dialyzed, and finally fractionated on a hydroxyapatite column with Tris-HCl buffer, pH 7, as shown in Fig. 1.
The assay system for the starch granule-bound enzyme contained 0.1 µmol. of (14C) GIc-1-P (500 cpm/nmol.), 0.4 µmol. of Mg2+, 5 mg of starch granules in a final volume of 60 µl of 0.125 M MES buffer, pH 6.0. After incubation, either the reaction mixture was centrifuged to remove the starch granules, and an aliquot of the supernatant fraction was placed in 0.5 ml (5mg) of sweet corn phytoglycogen solution, then precipitated with 1 ml of 95% ethanol; or to the reaction mixture, 0.5 ml (5mg) of sweet corn phytoglycogen solution was added (without removing the starch granules), then precipitated with 1 ml of 95% ethanol. The precipitate was collected and washed four times with a total of 4 ml of 95% ethanol before suspending in 0.5 ml of water for counting. The assay system for the solubilized enzyme contained 1 µmol. Of (14C) Glc-1-P (30 µl. 500 cpm/nmol.), and 100 µl of the hydroxyapatite column-purified enzyme in a final volume of 130 µl of 0.125 M MES buffer, pH 6.0 containing 1 mM DTT. After incubation, the reaction was stopped by the addition of 0.5 ml (5mg) of sweet corn phytoglycogen and precipitation with ethanol as described above for the starch granule-bound enzyme.
The present study shows that the reference mutation at the bt1 locus results in a deficiency of a starch granule-bound phospho-oligosaccharide synthase. The enzyme activity of hydroxyapatite column-purified enzymes from nonmutant and the bt1 mutant is shown in Fig. 1. The data show the phospho-oligosaccharide synthase activity in the developing endosperms of bt1 is remarkably low at this stage of development. Fig. 2 shows the enzyme activity of starch granule-bound phospho-oligosaccharide synthase measured at various post-pollination times in developing endosperms of nonmutant versus bt1 endosperms. The main point illustrated in this figure is that a similar developmental profile for both nonmutant and bt1 was observed; however, the enzyme activity of bt1 is significantly lower than the normal line, particularly when the data are expressed in terms of the number of starch granules used for assay (see inset of Fig. 2), rather than on equal weights of starch granules as is done in Figures 1 and 2. Moreover, we also found that there is no significant difference in the level of the soluble phospho-oligosaccharide synthase in the developing endosperms of either normal or bt1 (data not shown).
Thus, the results reported here show a marked decrease in the activity of a starch granule-bound phospho-oligosaccharide synthase as a consequence of the bt1 mutation although it has not yet been demonstrated that the deficiency is the primary biochemical lesion in this mutant.
David Pan and Oliver Nelson, Jr.
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