Molecular analysis of the effects of the lys 3a gene on the expression of Hor loci in developing endosperms of barley (Hordeum vulgare L.)

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Abstract
The lys 3a gene present in the barley mutant Risø 1508 results in an increased content of lysine in the grain. Previous studies have shown that this increase results from a decreased accumulation of hordein and an increase in other more lysine-rich proteins and in free amino acids. We report here a detailed examination of the effects of this gene on the different groups of hordein polypeptides and the mRNAs encoding them. The amounts of the two major groups of hordein polypeptides (B and C hordein) were reduced to about 20 and 7%, respectively, of those present in the parental variety (Bomi), with a greater effect on one of the two subfamilies of B polypeptides. In contrast, the amounts of D hordein polypeptides were increased fourfold. In vitro translations of polysomal and total cellular RNA fractions showed similar effects on the relative amounts of hordein products synthesized. More detailed analyses of the populations of hordein mRNAs were made using specific cDNA clones and hybrid-selection translation, Northern hybridization, and “hybrid-dot” analysis. Only traces of mRNAs for “C” hordein were detected, while the abundances of mRNAs for the two subfamilies of B hordeins were reduced to 40 and 5% of those in Bomi. The amount of mRNA for D hordein was increased twofold. A cDNA clone related to B hordein was used to analyze genomic DNA fractions by Southern hybridization. The lys 3a gene had no effect on either the number (about 10) or the organization of the B hordein genes. These studies clearly demonstrate that the effects of the lys 3a gene on the amounts of the hordein polypeptides are closely related to changes in the amounts of the mRNAs encoding them. Although the exact effect of the gene remains unknown, it is most likely to be either at transcription or on the early processing of the mRNA.