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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bantle, J. A., Maxwell, I. H., and Hahn, W. E. (1976). Specificity of oligo(dT) cellulose chromatography in the isolation of polyadenylated RNA. Anal. Biochem. 72413.
Brandt, A. (1976). Endosperm protein formation during kernal development of wild type and high lysine barley mutant. Cereal Chem. 53890.
Brandt, A., and Ingversen, J. (1976). In vitro synthesis of barley endosperm proteins on wild type and mutant templates. Carlsberg Res. Commun. 41313.
Brandt, A., and Ingversen, J. (1978). Isolation and translation of hordein messenger RNA from wild type and mutant endosperms in barley. Carlsberg Res. Commun. 43451.
Burr, F. A., and Burr, B. (1982). Three mutations in Zea mays affecting zein accumulation: A comparison of zein polypeptides, in vitro synthesis and processing, mRNA levels and genomic organization. J. Cell. Biol. 94201.
Chamberlain, J. P. (1979). Fluorographic detection of Radioactivity in polyacrylamide gels with the water soluble flour, sodium salicylate. Anal. Biochem. 98132.
Clewell, D. B. (1972). Nature of Col E1 plasmid replication in Escherichia coli in the presence of chloramphenicol. J. Bacteriol. 110667.
Doll, H. (1980). A nearly non-functional mutant allele of the storage protein locus Hor-2 in barley. Hereditas 93217.
Doll, H., Køic, B., and Eggum, B. O. (1974). Induced high lysine mutants in barley. Radiat. Bot. 1473.
Faulks, A. J., Shewry, P. R., and Miflin, B. J. (1981). The polymorphism and structural homology of storage proteins (hordein) coded for by the Hor-2 locus in barley (Hordeum vulgare L.). Biochem. Genet. 19841.
Field, J. M., Shewry, P. R., Miflin, B. J., and March, J. F. (1982). The purification and characterization of homologous high molecular weight storage proteins from grain of wheat, rye and barley. Theor. Appl. Genet. 62329.
Forde, B. G., Kreis, M., Bahramian, M. B., Matthews, J. A., Miflin, B. J., Thompson, R. D., Bartels, D., and Flavell, R. B. (1981). Molecular cloning and analysis of cDNA sequences derived from poly A+ RNA from barley endosperm: Identification of B hordein-related clones. Nucleic Acids Res. 96689.
Forde, J., Forde, B. G., Kreis, M., Fry, R., Shewry, P. R., and Miflin, B. J. (1983). FEBS Letts. 162360.
Fox, J. E., Pratt, H. M., Shewry, P. R., and Miflin, B. J. (1977). The in vitro synthesis of hordeins with polysomes from normal and high-lysine varieties of barley. In Nucleic Acids and Protein Synthesis in Plants CNRS, Paris, p. 520
Friedman, M., Krull, L. H., and Cavins, J. F. (1970). The chromatographic determination of cystine and cysteine residues in proteins as S-β-(4-pyridylethyl) cysteine. J. Biol. Chem. 2453868.
Grunstein, M., and Hogness, D. S. (1975). Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene. Proc. Natl. Acad. Sci. USA 733146.
Ingversen, J., Køie, B., and Doll, H. (1973). Induced seed protein mutant of barley. Experientia 291151.
Jensen, J. (1979). Chromosomal location of the dominant and four recessive high-lysine mutants. In Seed Protein Improvement in Cereals and Grain Legumes, Vol. 1 IAEA, Vienna, pp. 89–96.
Jensen, J., Jorgensen, J. H., Jensen, H. P., Giese, H., and Doll, H. (1980). Linkage of the Hordein loci Hor 1 and Hor 2 with the powdery mildew resistance loci Ml-k and Ml-a on Barley chromosome 5. Theor. Appl. Genet. 5827.
Kasha, K. J., and Kao, K. N. (1970). High frequency haploid production in barley (Hordeum vulgare L.) Nature 255874.
Kirkman, M. A., Shewry, P. R., and Miflin, B. J. (1982). The effect of nitrogen nutrition on the lysine content and protein composition of barley seeds. J. Sci. Food Agr. 33115.
Kreis, M. (1978). Starch and free sugars during kernel development of Bomi barley and its high-lysine mutant 1508. In Seed Protein Improvement by Nuclear Techniques IAEA, Vienna, pp. 115–120.
Kreis, M., and Doll, H. (1980). Starch and prolamin level in single and double high-lysine barley mutants. Physiol. Plant. 48139.
Kreis, M., Rahman, S., Forde, B. G., Pywell, J., Shewry, P. R., and Miflin, B. J. (1983a). Sub-families of hordein mRNA encoded at the Hor 2 locus of barley. Mol. Gen. Genet. 191194.
Kreis, M., Shewry, P. R., Forde, B. G., Rahman, S., and Miflin, B. J. (1983b). Molecular analysis of a mutation conferring the high-lysine phenotype on the grain of barley (Hordeum vulgare). Cell 34161.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277681.
Langridge, P., Pinto-toro, J. A., and Feix, G. (1982). Transcriptional effects of the opaque-2 mutation of Zea mays L. Planta 156166.
Matthews, J. A., and Miflin, B. J. (1980). In vitro synthesis of barley storage proteins. Planta 149262.
Miflin, B. J., and Shewry, P. R. (1979). The synthesis of proteins in normal and high-lysine barley seeds. In Laidman, D., and Wyn Jones, R. G. (eds.), Recent Advances in the Biochemistry of Cereals Academic Press, London, pp. 239–273.
Miflin, B. J., Rahman, S., Kreis, M., Forde, B. G., Blanco, L., and Shewry, P. R. (1983). The hordeins of barley: Developmentally and nutritionally regulated multigene families of storage proteins. In Ciferri, O., and Dure, L. S. (eds.), Structure and Function of Plant Genomes Plenum Press, New York pp. 85–92.
Munck, L., Karlsson, K. E., Hagberg, A., and Eggum, B. O. (1970). Gene for improved nutritional quality in barley. Science 168985.
Park, W. D., Lewis, E. D., and Rubenstein, I. (1980). Heterogenity of zein mRNA and protein in maize. Plant Physiol. 6598.
Rahman, S., Shewry, P. R., and Miflin, B. J. (1982). Differential protein accumulation during barley grain development. J. Exp. Bot. 33717.
Rahman, S., Kreis, M., Forde, B. G., Shewry, P. R., and Miflin, B. J. (1983a). Hordein mRNAs in developing barley grain (in preparation).
Rahman, S., Shewry, P. R., Forde, B. G., Kreis, M., and Miflin, B. J. (1983b). Nutritional control of storage protein synthesis in developing grain of barley (Hordeum vulgare L.) Planta 159366.
Ricciardi, R. P., Miller, J. S., and Roberts, B. E. (1979). Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation. Proc. Natl. Acad. Sci. 764927.
Salcedo, G., Sanchez-Monge, R., Argamentaria, A., and Aragoncillo, C. (1980). The A-hordeins as a group of salt soluble hydrophobic proteins. Plant Science Lett. 19109.
Sanchez, F., Campomanes, M., Quinto, C., Hansberg, W., Mora, J., and Palacios, R. (1978). Nitrogen source regulates glutamine synthetase mRNA levels in Neurospora crassa. J. Bacteriol. 136880.
Shewry, P. R., and Miflin, B. J. (1982). Genes for the storage proteins of barley. Qual. Plant. Plant Foods Hum. Nutr. 31251.
Shewry, P. R., Ellis, J. R. S., Pratt, H. M., and Miflin, B. J. (1978a). A comparison of methods for the extraction and separation of hordein fractions from 29 barley varieties. J. Sci. Food Agr. 29433.
Shewry, P. R., Kirkman, M. A., Pratt, H. M., and Miflin, B. J. (1978b). Storage protein formation in normal and high lysine barley. In Miflin, B. J., and Zoschke, M. (eds.), Carbohydrate and Proteins Synthesis EEC, Luxembourg, pp. 155–171.
Shewry, P. R., Pratt, H. M., and Miflin, B. J. (1978c). The varietal identification of single seeds of barley by analysis of hordein polypeptides. J. Sci. Food Agr. 29847.
Shewry, P. R., Pratt, H. M., Faulks, A. J., Parmar, S., and Miflin, B. J. (1979a). The storage protein (hordein) polypeptide pattern of barley in relation to varietal identification and disease resistance. J. Natl. Inst. Agr. Bot. 1534.
Shewry, P. R., Pratt, H. M., Leggatt, M. M., and Miflin, B. J. (1979b). Protein metabolism in developing endosperms of high-lysine and normal barley. Cereal. Chem. 56110.
Shewry, P. R., Faulks, A. J., and Miflin, B. J. (1980a). Effect of high lysine mutations on the protein fractions of barley grain. Biochem. Genet. 18133.
Shewry, P. R., Faulks, A. J., Pickering, R. A., Jones, I. T., Finch, R. A., and Miflin, B. J. (1980b). The genetic analysis of barley storage proteins. Heredity 44383.
Shewry, P. R., Field, J. M., Kirkman, M. A., Faulks, A. J., and Miflin, B. J. (1980c). The extractions, solubility and characterization of two groups of barley storage polypeptides. J. Exp. Bot. 121393.
Shewry, P. R., Lew, E. J.-L., and Kasarda, D. D. (1981). Structural homology of the storage proteins coded by the Hor-I locus of barley (Hordeum vulgare L.). Planta 153246.
Shewry, P. R., Finch, F. A., Parmar, S., Franklin, J., and Miflin, B. J. (1983a). Chromosomal location of Hor-3, a new locus governing storage proteins in barley. Heredity 50179.
Shewry, P. R., Franklin, J., Parmar, S., Smith, S. J., and Mifflin, B. J. (1983b). The effects of sulphur starvation on the amino acid and protein composition of cereal grain. J. Cereal Sci. 121.
Soave, C., Tardani, I., DiFonzo, N., and Salamini, F. (1981). Zein level in maize endosperm depends on a protein under control of the opaque-2 and opaque-6 loci. Cell 27403.
Thompson, R. D., and Bartels, D. (1983). Hordein messenger RNA levels in wild type and mutant barley endosperm. Plant Sci. Lett. (in press).
Author information
Authors and Affiliations
Additional information
This work was supported by EEC Grant 470.
Rights and permissions
About this article
Cite this article
Kreis, M., Shewry, P.R., Forde, B.G. et al. 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, 231–255 (1984). https://doi.org/10.1007/BF00484227
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00484227