Expression of the gamma-zein protein of maize in seeds of transgenic barley: effects on grain composition and properties.

A cDNA clone encoding the gamma-zein protein of maize was expressed in developing grain of barley using the starchy endosperm cell-specific promoter from the wheat Glu-1D-1 (HMW subunit 1Dx5) gene. Seven transgenic lines were recovered from 226 bombarded immature embryos, of which two were sterile and four tetraploid, while five were shown to express the gamma-zein protein based on western blotting. Southern blot analysis showed the presence of between about three and twelve transgene insertions. Detailed comparative studies of five null and five homozygous transformed sub-lines from transgenic line A showed that gamma-zein accounted for over 4% of the total prolamin fraction, corresponding to about 1.9% of the total grain N. Comparison of the proteins present in the gel protein fraction demonstrated that the gamma-zein was incorporated into polymers, as in maize. However, there was no effect on grain hardness measured using the Perten Single Kernel Characterisation System or on the vitreousness measured by visual inspection. This contrasts with the situation in maize where a clear association with vitreousness has been reported.

Identification and molecular characterisation of hordoindolines from barley grain.

Grain texture in barley is an important quality character as soft-textured cultivars have better malting quality. In wheat, texture is considered to be determined by the puroindolines, a group of basic hydrophobic proteins present on the surface of the starch granule. Hard wheats have been proposed to lack puroindoline a or to have mutant forms of puroindoline b which do not bind to the granule surface. Analysis of six barley cultivars (three soft-textured and three hard) showed that all contained proteins homologous to wheat puroindoline b, but PCR analysis failed to show any differences in amino acid sequences similar to those which have been proposed to determine textural differences in wheat. Southern blot analysis showed two hordoindoline b genes which were isolated and shown to encode proteins with 94% sequence identity. Expression of hordoindoline b mRNA occurred in the starchy endosperm and aleurone layer of the developing seed, but not in the embryo. Analysis of seven soft- and six hard-textured barley varieties showed that all contained hordoindoline a except two hard varieties (Sundance, Hart) which were subsequently shown to both lack hordoindoline a mRNA. It was therefore concluded that there is not a clear relationship between the presence of hordoindoline a and grain texture in barley.