University of Wisconsin
Identification of glucosidase-transferase enzyme in developing maize endosperms that affects starch synthesis in the glt1 mutant --Pan, D We report the identification of an enzyme in developing maize endosperms that has both alpha-1,6-glucosidase and transferase activities. The transferase transfers oligosaccharides forming largely alpha-1,4-glucan bonds, although we cannot exclude the possibility that some alpha-1,6 linkages are formed. The spectrum of the amylopectin component of glt1 starch following reaction with an I2, KI stain has its absorption peak displaced from that of glt1 amylopectin, while the spectra of the amylose components are identical. This result indicates that glucosidase-transferase function is likely linked to the amylopectin synthesis pathway rather than the amylose pathway. The Brabender amylograms reveal that the starch produced by mutant endosperms differs markedly from that produced by nonmutant endosperms. The enzyme is coded by the nonmutant alleles of a gene on the short arm of chromosome 4 that we are designating glucosidase-transferase1 (glt1). The homozygous mutant seeds (glt1/glt1) have deeply dimpled crowns, somewhat shrunken, and are easily distinguished from their nonmutant sibs segregating on the same ear (Fig.1). The mutant seed weight, on average, is only 81% as much as the nonmutant. The catalytic activities of this enzyme are reminiscent to some extent of the glycogen debranching enzyme from rabbit muscle investigated by Brown. The purest preparation of that enzyme retained both glucosidase and transferase activities, and it is concluded that both activities were the properties of a single protein. The molecular weight of the maize enzyme is about 89,000 on SDS electrophoresis gel. The observations that starch production is reduced in mutant seeds and the amylopectin component is altered indicate a role in starch synthesis for this enzyme in maize. The relationship of the number of nonmutant alleles per endosperm to enzyme activity was investigated in a gene dosage series (zero to three nonmutant alleles/endosperm) made by selfing the glt1/glt1 and Glt1/Glt1 stocks and also crossing them reciprocally. The endosperms from each genotype were assayed for alpha-1,6-glucosidase activity using partially purified enzyme preparations (20 to 50% ammonium sulfate fraction). Fig. 2 shows the results expressed as nmol maltose released per endosperm over a 100 min incubation period. Such a linear relationship between enzyme activity and gene dosage would be expected if the Glt1 alleles encoded the enzyme, although it does not constitute definitive evidence of such a relationship. A bifunctional enzyme of this type has been investigated in rabbit muscle where it was presumed to be involved in the catabolism of glycogen. The maize enzyme can be shown to differ in some specifics from the mammalian enzyme although it also is an indirect debranching enzyme as classified by Lee and Whelan since it does not attack long glucose chains originating at an alpha-1,6-branch point. The enzyme in maize evidently has a role in starch synthesis in the developing endosperm as shown by the reduction in starch content of mutant endosperms, as well as alteration of amylopectin component. Thus, it is therefore suggested that four enzymes: branching enzyme (Ae), isoamylase/debranching enzyme (Su1), and alpha-1,4-glucosidase-transferase (Glt1) together play an important role for the modification of starch structure/synthesis during the development of maize endosperms.

Figures 1 and 2.

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