Molecular cloning of two isoinhibitor forms of chymotrypsin inhibitor 1 (CI-1) from barley endosperm and their expression in normal and mutant barleys

Abstract

Full-length cDNA clones for barley chymotrypsin inhibitor 1 (CI-1) have been isolated from an endosperm-specific cDNA library. Hybridization and nucleotide sequence analyses indicate that these cDNAs represent two distinct types of CI-1 mRNA which we have called CI-1A and CI-1B. Both mRNAs encode polypeptides of 83 residues (M r=8790 and 8960) which differ at eleven positions. The full-length cDNA sequences do not predict N-terminal signal peptide extensions indicating that CI-1 is synthesized in the mature form in contrast to the homologous proteinase inhibitors of tomato and potato. Northern hybridization experiments show that the CI-1 genes are under strict developmental and organ-specific control. CI-1 transcripts were first detected in the developing barley endosperm between 12 and 14 days after anthesis but no CI-1-related sequences were detected in the RNA preparations from shoots, leaves or roots. The expression of CI-1 was also studied in the high-lysine barley mutants Hiproly, Risø 56 and Risø 1508. Approximately 15-fold (Hiproly) and 4-fold (Risø 56 and 1508) higher levels of CI-1 mRNA were detected in the mutant endosperms compared to normal barley. These results correlate well with the increased deposition of CI-1 in the high-lysine lines and show that the differential expression is controlled mainly at the level of transcription or stability of the mRNA. Using Southern-blots of barley DNA we estimate that there are three copies of CI-1 per haploid genome in both normal and mutant barley lines.

Methylation of B-hordein genes in barley endosperm is inversely correlated with gene activity and affected by the regulatory gene Lys3.

“…Although such a model is possible, the increased expression of these genes can easily be explained as a secondary effect possibly me- diated by the increased level of free amino acids in lys3a endosperm (33). This hypothesis is supported by the equally increased expression of chymotrypsin inhibitor 1 in the lys3a and hor2ca mutants (34), as the latter is a deletion of the structural genes encoding B-hordein (35). The genomic sequencing shows that lys3a is more probably involved in early events of barley endosperm differentiation because the absence or inhibition of a trans-acting factor that interacts at specific sites in the promoters of the affected genes could not cause the differential methylation observed throughout the B-hordein promoters.…”

Opportunities for manipulating the seed protein composition of wheat and barley in order to improve quality

“…1) and studied the populations of mRNAs in developing endosperms and other tissues of normal and high lysine lines (Williamson et al, , 1988. They have also isolated a gene for CI-2 (Peterson et al, 1991) in order to explore the control of its expression.…”

“…Note the high content of lysine residues (K) and absence of cysteine. Based on data in Williamson et al (1987Williamson et al ( , 1988. Standard single letter abbreviations for amino acids are used.…”

Multi-method research strategy for understanding changes in barley grain protein composition and its relation to improved nutritional quality

“…This suggestion is strongly supported by a direct comparison of these antisense C-hordein lines with the spontaneous high-lysine barley mutant Hiproly, which contains the single recessive allele lys1 and the hordein percentage was shown to be slighted reduced (MUNCK et al, 1970). Some authors attributed the high-lysine content of this mutant and its derivatives to an increase of lysine-rich proteins composing the salt soluble protein fraction, such as protein Z (whose expression is enhanced by lys1), β-amylase and chymotrypsin inhibitors (CI-1) and CI-2 (BRANDT, 1976;HEJGAARD;BOISEN, 1980;KREIS, 1988).…”

“…Nonetheless, some examples of successful modification, mainly regarded to multiple lysine substitution of CI-2 in order to improve nutritional quality of barley grains can be found in the literature (ROESLER; RAO, 1999;FORSYTH et al, 2005). In addition, as mentioned above, higher expression of CI-1 and CI-2 is associated with the high-lysine phenotype in barley (WILLIAMSON et al, 1987;KREIS, 1988).…”