Last year crosses of TB-3Sb were made to plants heterozygous for brown aleurone (brn), a brown kerneled, seedling lethal mutant (MNL 59:7, 1985). The ears segregated for small, brown, almost defective kernels with brown embryos, and plump yellow kernels with shriveled, brown germs. It was suggested that the brown "defective" kernels had hypoploid endosperms and hyperploid embryos, while the yellow, near-germless kernels had hypoploid embryos and hyperploid endosperms. The defectiveness of the endosperm and embryo in these two seed types, respectively, were suggested to be due to hemizygosity at the brn locus.
In order to test these ideas, seeds of both types were planted this past summer. The yellow near-germless seeds did not germinate, but the brown defective kernels did, and the putative hyperploid plants were selfed and outcrossed onto cl1cl1Cl3Cl3 silks to test for the presence of TB-3Sb. The selfed ears segregated for brn, but in a reduced ratio expected of hyperploid plants. The outcross ears segregated for the pale yellow kernels expected of cl1. These results confirm the hyperploid status of the male parent, and the location of brn on the short arm of chromosome 3.
Since the brown defective kernels were shown to have hyperploid embryos, the brown pigmentation of these embryos must be due to diffusion of pigment from the endosperm. That the presence of the brown pigment in itself does not result in seedling lethality is shown by the viability of these kernels. The seedling lethality seen in homozygous brn plants must be due to other factors, possibly the absence of an essential gene product, resulting in blockage of a metabolic pathway. The brown pigment may represent a precursor that accumulates behind such a block, or perhaps a degradation product of such a precursor.
Several attempts to extract the brown pigment from finely ground kernels were made, using a broad range of solvents. Standard procedures using hexane and acetone to extract carotenoids (F.P. Zscheile and J.W. Porter, Analytical Chemistry 19:47-51, 1947) removed carotenoid pigments, but left the brown pigment intact. Likewise, solutions of 100% and 90% methanol, and 70% ethanol, removed remaining carotenoids, and presumably any flavonoids that were present, but left the brown pigment. Solvents that partially extracted the pigment include .1N aqueous HCl and .5M NaCl. A buffer containing 5% 2-mercaptoethanol and 2.3% SDS seemed to remove most of the remaining pigment. These observations suggest that the brown pigment is not a carotenoid or an anthocyanin. Its extraction by solvents used in the extraction of proteins (.5M NaCl and SDS buffer) suggests that it could be a protein, a protein degradation product, or an unrelated compound that is tightly bound by or associated with protein. These possibilities, and others, will be further investigated.
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