A comparison was made of the effectiveness of various solvents in extracting protein from wheat gluten. The best proved to be 0 . 0 5 5~ cetyltrimethyl ammonium bromide-6~ urea-0.0lh.I acetic acid. When extracts were separated on columns of controlledpore glass, with a nominal exclusion limit of 1 . 2~ lo6 daltons for molecules in the extended form, two peaks were obtained termed F1 and F2. F1 was excluded from the column and contained material which was stable in the absence of reducing agent but was markedly reduced in size in the presence of 2-mercaptoethanol. SDS-PAGE showed that F1 was enriched in high molecular weight polypeptides (about 100000 daltons) and also contained polypeptides with molecular weights in the range 30000-45000. F2 consisted almost entirely of polypeptides with molecular weights of less than 50000. The ratio of F1 :F2 in different varieties varied from 0.1 to 0.58 and this variation was correlated with the NIAB potential breadmaking quality score. However, there did not appear to be any difference between the total amounts of high molecular weight polypeptides in a poor and a good breadmaking variety.
cDNA clones encoding dioscorins, the major tuber storage proteins (M(r) 32,000) of yam (Dioscorea cayenesis) have been isolated. Two classes of clone (A and B, based on hybrid release translation product sizes and nucleotide sequence differences) which are 84.1% similar in their protein coding regions, were identified. The protein encoded by the open reading frame of the class A cDNA insert is of M(r) 30,015. The difference in observed and calculated molecular mass might be attributed to glycosylation. Nucleotide sequencing and in vitro transcription/translation suggest that the class A dioscorin proteins are synthesised with signal peptides of 18 amino acid residues which are cleaved from the mature peptide. The class A and class B proteins are 69.6% similar with respect to each other, but show no sequence identity with other plant proteins or with the major tuber storage proteins of potato (patatin) or sweet potato (sporamin). Storage protein gene expression was restricted to developing tubers and was not induced by growth conditions known to induce expression of tuber storage protein genes in other plant species. The codon usage of the dioscorin genes suggests that the Dioscoreaceae are more closely related to dicotyledonous than to monocotyledonous plants.
Two approaches were used to study the aggregation states of prolamins extracted from milled whole grain of wheat, barley, rye and maize with 500 ml litre-I aqueous propan-1-01. In the first, a comparison was made of the electrophoretic patterns of the samples separated by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions. In the second, they were chromatographed on a column of controlled pore glass (CPG) using a modified acetic acid, urea, cetyltrimethyl ammonium bromide (AUC) solvent and aliquots of the major peaks were reduced and analysed by SDS-PAGE. When the CPG chromatography was repeated under reducing conditions some of the peaks were eliminated indicating that they were composed of disulphide-linked aggregates. The results from both approaches showed that in barley, rye and wheat the HMW prolamins and some of the S-rich prolamins (B hordein, some y-secalins and subunits of high molecular weight gliadins) are present predominantly or partially in high molecular weight (above 1 x 106 daltons) aggregates which appear to be stabilised by disulphide bonds.Other S-rich prolamins (including some y-secalins, a, p and y-gliadins) and all the Spoor prolamins ('C' hordein, w-secalin, w-gliadin) are present predominantly or only as monomers. These results are discussed in relation to the structural and genetic homology of the prolamins in the three species. Although prolamin aggregates are also present in maize, these are of lower molecular weight.
Understanding iron trafficking in plants is key to enhancing the nutritional quality of crops. Due to the difficulty of imaging iron in transit, little is known about iron translocation and distribution in developing seeds. A novel approach, combining 57Fe isotope labelling and NanoSIMS, was used to visualize iron translocation dynamics at the subcellular level in wheat grain, Triticum aestivum L. We were able to track the main route of iron from maternal tissues to the embryo through different cell types. Further evidence for this route was provided by genetically diverting iron into storage vacuoles, as confirmed by histological staining and TEM-EDS. Virtually all iron was found in intracellular bodies, indicating symplastic rather than apoplastic transport. Aleurone cells contained a new type of iron body, highly enriched in 57Fe, and most likely represents iron-nicotianamine being delivered to phytate globoids. Correlation with tissue-specific gene expression provides an updated model of iron homeostasis in cereal grains with relevance for future biofortification efforts.
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