Cloning of a wheat 15‐kDa grain softness protein (GSP)

Abstract

The wheat starch 15-kDa protein (called grain softness protein or GSP) consists of a major polypeptide and several minor polypeptides. An antiserum raised against GSP was used to screen a wheat cDNA library. A cDNA family encoding approximately 15-kDa proteins that included a heptapeptide sequence previously isolated from protease digests of GSP was identified. A partial cDNA was used in a prokaryotic expression system to produce a fusion protein which reacted strongly against the original anti-GSP serum. A new antiserum raised against the fusion protein produced a weak reaction against a 15-kDa polypeptide extracted from wheat seeds. The results suggest that the proteins encoded by the cDNA family form a minor component of the mixture of 15-kDa polypeptides defined as GSP. RNA complementary to the cDNAs could be extracted from both soft and hard wheat grains from about half-way through grain filling. The encoded proteins are novel members of the 2s superfamily of seed proteins, a diverse family of proteins which maintain a characteristic framework of cysteine residues. The deduced proteins show the highest similarity to the oat 16-kDa avenin and to wheat puroindoline (a lipid-binding 15-kDa protein from wheat). Review of previously published data shows that puroindoline is also closely related to the major polypeptide of GSP, suggesting that the lipid-binding properties of GSP polypeptides may influence grain softness.

Sequencing of the Triticum monococcum Hardness locus reveals good microcolinearity with rice

“…It is a simply inherited character (Symes 1965) and although the main locus is referred as ''Hardness'', softness is in fact the dominant trait. Soft wheat varieties carrying the dominant Ha allele have a higher abundance of a starch surface-associated protein (M r =15 kDa) named friabilin (Greenwell and Schofield 1986;Rahman et al 1994). Friabilin is a composite of related lipid-binding proteins including proteins coded by the genes Puroindoline-a (Pina) and Puroindoline-b (Pinb) and, in lower proportion, the protein coded by the Grain Softness Related (Gsp) gene (Gautier et al 1994;Rahman et al 1994).…”

“…Soft wheat varieties carrying the dominant Ha allele have a higher abundance of a starch surface-associated protein (M r =15 kDa) named friabilin (Greenwell and Schofield 1986;Rahman et al 1994). Friabilin is a composite of related lipid-binding proteins including proteins coded by the genes Puroindoline-a (Pina) and Puroindoline-b (Pinb) and, in lower proportion, the protein coded by the Grain Softness Related (Gsp) gene (Gautier et al 1994;Rahman et al 1994). The puroindoline proteins are more similar to each other (71%) than to the Grain Softness Related Protein (Gsp, 57-58%).…”

Genetic and physical characterization of grain texture-related loci in diploid wheat

“…Speci®c PCR primers were designed based on the sequences of pTa31 (Pina-D1; Gautier et al 1994) and pGsp clones (Rahman et al 1994). The sense-and antisense-strand primers for the Pina-1 gene were 5¢-CCCTGTAGAGACAAAGCTAA-3¢ and 5¢-TCAC-CAGTAATAGCCAATAGTG-3¢; and for the Gsp-1 gene were 5¢-GCAAGCTCCCACCGCGGATG-3¢ and 5¢-CCAGTAATAT-CCGCTAGTGAT-3¢.…”

“…Clones for the Ha-related genes Clone pGsp for the grain softness-related protein was kindly supplied by S. Rahman (Rahman et al 1994). Clone pTa31, which corresponds to the Pina-D1 cDNA, was kindly provided by P. Joudrier (Sourdille et al 1996).…”

“…cDNAs encoding for these proteins have been characterized (Gautier et al 1994;Rahman et al 1994) and mapped on the distal part of chromosome 5DS completely linked to the Ha locus. The Pina-1 and Pinb-1 loci have been detected only on the 5D chromosome but homoeologous Gsp-1 loci were detected on chromosomes 5A, 5B and 5D (Dubcovsky and Dvorak 1995;Jolly et al 1996;Sourdille et al 1996;Giroux and Morris 1997).…”