II. 23. The effects of mutant high lysine genes on the amount and composition of hordein.
P.R. Shewry, S. Parmar and J. Franklin, Rothamsted Experimental Station, Harpenden, Herts. AL5 2JQ, U.K. "R"
Hordein consists of three mayor groups of polypeptides (B, C and D hordeins), each encoded by a single multigenic locus (designated Hor 2, Hor 1, and Hor 3 respectively) located on chromosome 5 (see Shewry et al., 1983a). The Risø series of high lysine mutants all result in decreased accumulation of total hordein (see Doll, 1983). In addition, some have been reported to have effects on the relative amounts of B, C and D hordeins, or specific components of these (Koie and Doll, 1979; Kreis et al., 1984). To study these effects in more detail we have analysed hordein fractions from four Risø mutants and six double mutant combinations.
Seeds were supplied by Dr. H. Doll (Risø, Denmark) and were multiplied in the glasshouse at Rothamsted. Hordein fractions were extracted and analysed by SDS-PAGE and gel scanning (Shewry et al., 1983b). The results are summarized in Fig. 1.
Figure 1. The effects of mutant high lysine genes on the hordein fraction of barley grain.
The lower part of the figure shows SDS-PAGE separations of the fractions. B1, B2 and B3 are bands present in Bomi and other lines with the Hor 2 Ze allele. B1 and B2 + B3 correspond to the products of two sub-families of mRNA’s and presumable genes at the Hor 2 locus (Kreis et al., 1983). Quantitative determinations of the proportions of the groups of polypeptides are shown in the central part of the figure. In this B3 includes the B2 + B3 bands. The Hor 2 alleles in the Risø 29/Risø 1508 lines were determined by back-crossing.
The hordein patterns of the four single mutants are consistent with previous reports (Koie and Doll, 1979; Kries et al. 1984). Risø 7 and 527L were both induced in Bomi and have increased proportions of the B1 hordein band which is characteristic of the Hor 2 Ze allele. Risø 1508, also induced in Bomi, has drastic effects on C and B1 hordeins, less effect on B3 hordeins and little or no effect on D hordein. Risø 29, induced in Carlsberg II, affects the proportions of the B hordein bands encoded by the Hor 2 Ca allele.
The combinations of mutant genes have various effects on the hordein fractions, but certain points can be noted.
1. All double mutants with Risø 1508 have less hordein than Risø 1508 itself. The Risø 29/Risø 1508 lines have similar hordein patterns to Risø 1508, with minor differences in one line due to the presence of the Hor 2 Ca allele. The Risø 7/Risø 1508 lines have different patterns to Risø 1508 and to each other. The latter is surprising since these should be isogenic. The Risø 527/ Risø 1508 lines have almost no detectable B, C or D hordeins, although Risø 527 has only a small effect on the amount of hordein when alone or combined with Risø 7.
2. Risø 527 causes a specific decrease in D hordein, when alone or in combination.
3. Although Risø 7 and Risø 527 both result in an increased proportion of B1 hordein, this effect is not observed in the Risø 7/Risø 527 double mutant lines. In contrast an increased proportion of B1 hordein is present in the Risø 7/Risø 29 line with the Hor 2 Ze allele.
The results cannot be readily explained with our limited knowledge of hordein gene regulation. However, they are consistent with our previous observations that although Hor 1, Hor 2 and Hor 3 are under generally co-ordinated control, their relative levels of expression may be modulated by nutrition (Rahman et al., 1983), development (Rahman et al.,1984) and regulatory genes (Kreis et al., 1984).
References:
Doll, H. 1983, Barley seed proteins and possibilities for their improvement. In: Seed Proteins. Eds. W. Gottschalk and H. P. Muller. Martinus Nijhoff. The Hague, pp.207-223.
Koie, B. and Doll, H. 1979. Protein and carbohydrate components in the Risø high-lysine barley mutants. In: Seed Protein Improvement in Cereals and Grain Legumes. Vol. 1, IAEA, Vienna, p. 205-215.
Kreis, M., Rahman, S. Forde, B.G., Pywell, J., Shewry, P.R. and Miflin, B.J. 1983. Sub-families of hordein mRNA encoded at the Hor 2 locus of barley. Mol. Gen. Genet. 191:194.
Kreis, M., Shewry, P.R., Forde, B.G. Rahman, S. Bahramian, M.B. and Miflin, B.J. 1984. Molecular analysis of the effects of the lys 3a gene on the expression of Hor loci in developing endosperms of barley (Hordeum vulgare L.). Biochem. Genet. 22:231.
Rahman, S., Kreis, M., Forde, B. G., Shewry, P.R. and Miflin, B.J. 1984. Hordein gene expression during the development of the barley (Hordeum vulgare) endosperm. Biochem. J. 223: 351 .
Rahman, S., Shewry, P.R., Forde, B.G., Kreis, M., Miflin, B.J. 1983. Nutritional control of storage protein synthesis in developing grain of barley (Hordeum vulgare). Planta 159: 366.
Shewry, P.R., Finch, R., Parmar, S., Franklin, J. and Miflin, B.J. 1983a. Chromosomal location of Hor 3, a new locus governing storage proteins in barley. Heredity 50:179.
Shewry, P.R., Franklin, J., Parmar, S., Smith, S.J. and Miflin, B.J.
1983b. The effects of sulphur starvation on the amino acid and protein
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