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

Lead Research Organisation: Rothamsted Research
Department Name: Plant Biology & Crop Science

Abstract

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 microarray gene expression 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.

Technical Summary

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.

Publications

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Description Xylans are key components of plant cell walls and one of the most abundant biopolymers on earth. They differ in grasses from other plants (grasses include the most important food crops, wheat, rice and maize, as well as pasture species).The key difference of grass xylan is the addition of arabinose to the polymer backbone. In the work in this grant, we have discovered that wheat and rice genes in the GT61 gene family are responsible for this step. This breakthrough was published in PNAS and has been added to the faculty of 1000 of papers in top 2% of importance.
Exploitation Route As a result of the research in this grant, we expect wheat varieties to be developed in the next few years with improved properties. Wheat varieties used for non-food will have fewer problems associated with viscosity. The research will also accelerate the development of wheat varieties with high dietary fibre with improved health benefits for food. In the longer term, improved varieties of grass crops for biofuel and for ruminant nutrtion are likely. 1. Since we have discovered genes which alter the structural properties of arabinoxylan (AX) in wheat grain, we can tailor these genes to different end uses of wheat grain. For non-food uses, it is desirable to decrease soluble AX and this is subject of a patent application. For food uses, we intend to increase AX (dietary fibre) using natural variation.

2. Xylan is key to biomass digestibility. Therefore, the genes we have discovered are likely to prove important targets for increasing digestibilty of grass biomass for biofuel and ruminant feed.
Sectors Agriculture, Food and Drink

 
Description Yes. Genes identified have been used in follow-on project BB/K010824/1 to develop new wheat varieties carrying novel alleles in these. Also knowledge is used in identification of alleles underlying QTLs for high dietary fibre in collaboration with industry.
First Year Of Impact 2014
Sector Agriculture, Food and Drink
Impact Types Economic

 
Title GT43, GT47 or GT61 Silencing To Reduce Arabinoxylan In Wheat Grain 
Description We discovered the genes responsible for synthesising water-extractable arabinoxylan (WE-AX) in wheat grain. We recognised potential market for UK wheat variety where activity of these genes was decreased for non-food use, particularly Scotch Whisky and filed patent application GB1106964.8. We showed using transgenic approaches that suppressing these genes in with GM approach greatly decreased extract viscosity, a negative trait for non-food uses of wheat grain. We then pursued the same outcome using non-GM TILLING approach in follow-on and Agritech grants. Patent has now been awarded (August 2018). 
IP Reference WO2012143520 
Protection Patent granted
Year Protection Granted 2018
Licensed Commercial In Confidence
Impact Collaboration with wheat breeding company Limagrain which has led to follow-on award and Agrticech awards.