A proteomic approach to the identification and characterisation of protein composition in wheat germ.

Proteome analyses were carried out on commercial wheat germ of mature grain from the biscuit-making wheat cultivar, Rosella. Wheat germ protein extracts were fractionated by two-dimensional gel electrophoresis across two different immobilised pH gradients: pH 4.0-7.0 and 6.0-9.0. A total of 612 individual protein spots were excised from the gels and characterised by peptide mass fingerprinting. From these analyses, 347 individual proteins were identified from protein sequence database interrogation, and 301 different types of protein were catalogued according to protein function. The remaining 265 protein spots gave poor or no matches to proteins in the databases and were not identified in this study. Six different classes of enzymes were identified in the germ, many of them having roles in the mobilisation of energy reserves for germination. Abundantly expressed enzyme classes include the oxidoreductases, transferases and hydrolases. A comparison was also made between the major protein classes expressed in the germ and protein classes expressed in the endosperm from previous proteomic work. This study contributes significantly to our knowledge of protein expression and heterogeneity in the germ of wheat grain and forms the basis for future studies in regard to the characterisation of proteins during the initial stages of germination.

Black Point is associated with reduced levels of stress, disease- and defence-related proteins in wheat grain.

SUMMARY Black Point in wheat is a dark discoloration at the embryo end of the grain, which causes substantial financial losses to wheat growers due to down-grading of otherwise high-grade wheat. There does not appear to be a single cause for Black Point, although evidence suggests that fungal infection is the main link to Black Point symptoms. We sought to identify grain proteins from Black Point-affected and Black Point-free wheat cultivar SUN239V, which is known to be very susceptible to Black Point. The proteomes of both the germ and endosperm-bran components of Black Point-affected and Black Point-free grain were compared using two-dimensional gel electrophoresis (2-DE) with six replicate gels run for each protein sample. Approximately 1478 discrete protein spots were found in 2-DE gels from the germ fraction of the grain, of which 354 were identified by mass spectrometry (MS). Similarly, 1360 discrete protein spots were found from the endosperm-bran fraction, of which 303 were identified by MS. No proteins of fungal or bacterial origin were positively identified, suggesting that, at least in some cases, Black Point is not associated with microbial activity. Of the germ proteins, 252 were differentially expressed in Black Point-affected tissue, with 67 of these proteins identified by MS. Of the endosperm-bran proteins, 317 were differentially expressed in Black Point-affected tissue, with 86 identified. The largest of 12 functional classes to which the differentially abundant proteins were assigned was the 'stress' class, i.e. products of genes associated with stress, disease and defence. Higher levels of these proteins were found in Black Point-free grain, suggesting that protection from the disease might be afforded by increased levels of the 'stress' proteins.

Contrasting effects of chronic heat stress and heat shock on kernel weight and flour quality in wheat.

Phytotron studies were conducted to compare the potential effects of chronic high-temperatures (daily maxima of over 20˚C) and heat-shock conditions (a few days of over 32˚C), on wheat (Triticum aestivum L.) yield and quality, to form a basis for the selection of improved high-temperature tolerance in wheat. The series of heat-shock treatments were designed to provide similar heat loads, by varying the duration (number of days) of each treatment. Studies involved two cultivars, Lyallpur and Trigo 1. Both showed a reduction in kernel weight in response to chronic high day temperatures (i.e. above 18˚C), with Trigo 1 more tolerant than Lyallpur. Kernel weight of both cultivars was also reduced by short periods of heat shock, and this was most evident at day/night temperatures above 30/25˚C. There was no reduction in the germination of the lighter weight kernels formed under either chronic high temperature or heat-shock conditions. Dough strength, as judged by mixing time, declined in both genotypes with prolonged chronic high temperature, and also following the most extreme of the heat-shock treatments - Trigo 1 showed an ability to resist these changes better than Lyallpur. The heat-related decreases in dough strength were associated with decreases in the proportion of the larger molecular size glutenin (most 'unextractable'). This change in quality was not however, associated with changes in flour protein content.