RNAi suppression of xylan synthase genes in wheat starchy endosperm.

Mark D Wilkinson,Jessica Evans,Jane L Ward,Ondrej Kosik,Abigail J Wood,Hannah Walpole,Alison Lovegrove,Peter R Shewry,Kirstie Halsey,Rowan A C Mitchell

doi:10.1371/journal.pone.0256350

Abstract

The xylan backbone of arabinoxylan (AX), the major cell wall polysaccharide in the wheat starchy endosperm, is synthesised by xylan synthase which is a complex of three subunits encoded by the GT43_1, GT43_2 and GT47_2 genes. RNAi knock-down of either GT43_1 or all three genes (triple lines) resulted in decreased AX measured by digestion with endoxylanase (to 33 and 34.9% of the controls) and by monosaccharide analysis (to 45.9% and 47.4% of the controls) with greater effects on the amount of water-extractable AX (to 20.6 and 19.9% of the controls). Both sets of RNAi lines also had greater decreases in the amounts of substituted oligosaccharides released by digestion of AX with endoxylanase than in fragments derived only from the xylan backbone. Although the GT43_1 and triple lines had similar effects on AX they did differ in their contents of soluble sugars (increased in triple only) and on grain size (decreased in triple only). Both sets of transgenic lines had decreased grain hardness, indicating effects on cell wall mechanics. These results, and previously published studies of RNAi suppression of GT43_2 and GT47_2 and of a triple mutant of GT43_2, are consistent with the model of xylan synthase comprising three subunits one of which (GT47_2) is responsible for catalysis with the other two subunits being required for correct functioning but indicate that separate xylan synthase complexes may be responsible for the synthesis of populations of AX which differ in their structure and solubility.

Highlights

Cereal grains and products are the major sources of dietary fibre in the human diet
RNAi down-regulation of the single xylan synthase subunit encoded by GT43_1 and simultaneous down-regulation of all three subunits resulted in similar decreases in total AX, to 46% and 47% of the total X determined by monosaccharide analysis (Table 2). This compares with previously reported RNAi down-regulation of single xylan synthase subunit genes (GT43_2 or GT47_2) which resulted in decreases in total AX [10] to 50–60% of those in the control lines and the development of a triple knock-out mutant of GT43_2 in which total AX was reduced to about 65% of that in the control line [15]
The proportions of short Arabinoxylan Oligosaccharide (AXOS) released by GH11 digestion were decreased more in the single GT43_1 and triple lines lines than total AX, as was the proportion of WE-X (Table 2)

Summary

Introduction

Cereal grains and products are the major sources of dietary fibre in the human diet. Grain fibre, and the fibre present in whole grain, has well-established health benefits in reducing the risk of chronic diseases and certain forms of cancer [1]. Whereas whole wheat grains contain between about 10% and 15% fibre (dry weight basis) [2] most foods are made from white flour which is derived from the starchy endosperm of the grain and has a much lower fibre content (up to about 5%) [3]. Increasing the fibre content of white flour could have significant benefits for human health.

Methods

Results

Conclusion