Manipulation of cell wall synthesis to improve the dietary fibre composition of wheat flour

Manipulation of cell wall synthesis to improve the dietary fibre composition of wheat flour: Summary Wheat white flour products play a huge role in the human diet. The manifold health benefits of fibre in foods are now well established, but the useful fibre content of wheat flour is low compared to oats and barley. This is because the major wheat flour fibre component, arabinoxylan (AX) from the endosperm cell walls, is typically only 25% soluble and it is believed to be soluble fibre that confers the most health benefits. Thus increasing the soluble fibre content of wheat flour is a major target for public health improvement. The solubility of AX is determined by its structure; greater substitution of the xylan backbone by arabinose increases solubility, but cross-linked ferulic acids attached to these arabinose units decrease solubility. We recently identified candidate genes responsible for all the key steps in the synthesis of AX including the addition of arabinose residues and feruloylation using a novel bioinformatics approach. We have now augmented this with analysis of transcriptome data from developing wheat grain. This in general supports our published analysis (but has caused us to revise our view on the best candidate genes encoding arabinosyl transferases) so that we have strong candidates genes for these key steps which determine the solubility of AX. The proposed research is designed (1) to provide unequivocal evidence of the function of the enzymes (2) to demonstrate that manipulation of the encoding genes in transgenic wheat has the predicted effect on the amount of soluble AX in endosperm cell walls (3) to identify changed forms of the candidate genes in a mutant wheat population which are also predicted to increase the solubility of AX. The plants carrying this form of the genes are non-GM so can be used to develop commercial wheat varieties with increased soluble fibre (4) to map the genes so that molecular markers can be found for any wheat populations which show variation in these genes. This allows wheat breeders to rapidly incorporate any beneficial versions of the genes which exist naturally in populations into commercial varieties.

We intend to identify the genes and enzymes responsible for catalysing the synthesis of arabinoxylan (AX), focusing on xylan synthase, xylan arabinosyl transferase and xylan feruloyl transferase. We will use this knowledge to develop new wheat genotypes with enhanced soluble fibre composition of flour to improve its nutritional properties. The project can be divided into four parts: (1) Functional characterisation of candidate genes encoding enzymes of AX synthesis: genes which are predicted to encode enzymes for grass-specific steps will be expressed in Arabidopsis. The secondary cell walls of transformed lines will be analysed for the novel structrual features which would demonstrate gain-of-function using highly sensitive techniques (MS, PACE and GC-MS). In a second approach, insect cells will be transformed to express the enzymes. Secreted proteins will be used for in vitro assays using labelled acceptor or donor molecules and product detection by a combination of HPLC, PACE, MS techniques. (2) Demonstration of predicted effects of changes in gene expression on wheat dietary fibre: transgenic wheat lines will be generated which over-express putative arabinosyl transferase genes and have RNAi-induced decreased expression of feruloyl transferase and xylan synthase genes. Seed of these lines will be tested for the predicted increase in soluble AX in endosperm cells. (3) Identification of knock-down mutants of feruloyl transferase in a wheat TILLING population. Again the seed of these lines will be tested for increase in soluble AX. If successful, these lines will provide a non-GM route to increase dietary fibre content of wheat flour. (4) Map the characterized genes in hexaploid wheat using doubled-haploid populations and relate the loci to QTLs for soluble fibre content and composition. This will assist breeders to introduce beneficial alleles of these genes into commercial varieties.