Down-Regulation of the CSLF6 Gene Results in Decreased (1,3;1,4)-β-d-Glucan in Endosperm of Wheat

Abstract

(1,3;1,4)-b-D-Glucan (b-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative b-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total b-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable b-glucan was also reduced by about 50% in the transgenic lines, and the M r distribution of the fraction was decreased from an average of 79 to 85 3 10 4 g/mol in the controls and 36 to 57 3 10 4 g/mol in the transgenics. Immunolocalization of b-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a b-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.Cell wall polysaccharides account for about 10% of the dry weight of the mature wheat (Triticum aestivum) grain, and about 2% to 3% dry weight of the white flour fraction that is derived from the major storage tissue of the grain, the starchy endosperm (Stone, 1996). Although they are relatively minor components of white flour, the cell wall polysaccharides are immensely important in determining the properties of the flour for processing (Saulnier et al., 2007a) and in human nutrition, forming a major source of dietary fiber (Saulnier et al., 2007b;Topping, 2007).Wheat endosperm cell walls comprise two major components, arabino-(1,4)-b-D-xylan (arabinoxylan [AX]) and (1,3;1,4)-b-D-glucan (b-glucan), which account for about 70% and 20% of the total, respectively (Bacic and Stone, 1980). In addition about 4% cellulose [(1,4)-b-D-glucan] and 7% (1,4)-b-D-glucomannans are present (Bacic and Stone, 1980). This contrasts with starchy endosperm tissues of oats (Avena sativa) and barley (Hordeum vulgare), in which the proportions of AX and b-glucan are reversed (Henry, 1987;Stone, 1996). This is of particular interest as soluble b-glucans from barley and oats have benefits in reducing coronary heart disease that have been accepted by the U.S. Food and Drug Administration for health claims on food products (Anonymous, 2008). It is not known whether these benefits are shared by b-glucan from wheat, which differs from barley and oat b-glucans in its distribution of 1,3 and 1,4 lin...

RNA Interference Suppression of Genes in Glycosyl Transferase Families 43 and 47 in Wheat Starchy Endosperm Causes Large Decreases in Arabinoxylan Content

“…AX structure was characterized using simultaneous digestion of NSP preparations of white flour by xylanase and lichenase and analysis of the resulting oligosaccharides as described previously Nemeth et al, 2010;Pellny et al, 2012). Large decreases (significant at P , 0.001) were observed in the abundances of AXOS in all of the transgenic lines (Fig.…”

“…Although the HMW1Dx5 promoter drives strong expression at 10 DPA under our standard conditions , we have previously observed that the effects of RNAi transgenes driven by this promoter on cell walls do not become evident until 14 DPA (Nemeth et al, 2010); this delay is expected due to the time required for the RNAi machinery to operate and for existing enzymes to turn over. Here, we observed complete abolition of LM11 labeling in most of the starchy endosperm by 18 DPA in the TaGT47_2 line and a similar effect in the most distal layers of the dorsal region and of the lobes in the TaGT43_2 line (Fig.…”

“…Bread wheat (Triticum aestivum 'Cadenza') plants were grown in temperaturecontrolled glasshouse rooms as described (Nemeth et al, 2010).…”

“…Using a strong starchy endosperm-specific promoter to drive RNA interference (RNAi) to suppress specific transcripts, we have demonstrated the key role of TaCSLF6 in synthesizing 1,3;1,4-b-D-glucan (Nemeth et al, 2010) and that the GT61 gene TaXAT1 is responsible for nearly all the monosubstituted Ara in AX in endosperm (Anders et al, 2012). The amount and structure of AX in wheat starchy endosperm are also of great practical importance; as it is the main component of dietary fiber in wheat foods, it is a major contributor of dietary fiber in the human diet (Topping, 2007), and it also affects the processing properties of wheat flour for different end uses (Saulnier et al, 2007).…”

Pattern of Deposition of Cell Wall Polysaccharides and Transcript Abundance of Related Cell Wall Synthesis Genes during Differentiation in Barley Endosperm

“…Indeed, the barley blg [(1,3;1,4)-b-D-glucan less] mutant that has a mutation within the HvCSLF6 gene lacks (1→3, 1→4)-b-D-glucan in both endosperm and aleurone walls (Tonooka et al, 2009), confirming that this is the major gene contributing to (1→3, 1→4)-b-D-glucan content within the grain. Reduction of CSLF6 gene expression in wheat using RNA interference constructs has shown that this gene is also a major contributor to wheat endosperm cell wall (1→3, 1→4)-b-D-glucan (Nemeth et al, 2010).…”