Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat

Abstract

(1,3;1,4)-beta-d-Glucan (beta-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 beta-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 beta-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 beta-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 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-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 beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Figure 1.

Transcript abundance of CSL genes determined on the wheat Affymetrix GeneChip. Labels indicate the closest rice CSL gene that the transcript corresponds to; there are two wheat paralogs matching the rice OsCSLA7 gene. A, Whole-grain samples isolated at 10 stages of development in cv Hereward (each point is the average of two replicates; ANOVA of stages × genes gives a lsd 5% of 30.1 and there are significant differences between points at P < 0.001). B, Endosperm-enriched samples isolated at 14 to 21 DPA in cv Cadenza (each bar is the average of four replicates; ANOVA of genes gives a lsd 5% of 99.3 and there are significant differences between points at P = 0.001).

Figure 2.

Comparison of transcript abundance in line 15 transgenic (15T) and null segregant (N) samples of TaCSLF6 endogenous gene plus RNAi transgene (A) and TaCSLF6 endogenous gene only (B), determined by qRT-PCR using endosperm samples at 14 DPA. Bars represent average ± 1 se of ratio from four biological replicate samples for 15T and either four (A) or seven (B) biological replicate samples for nulls. [See online article for color version of this figure.]

Figure 3.

HPAEC analysis of oligosaccharides produced by enzyme mapping of transgenic and control lines. A trace from transgenic line 9 (green) is overlaid on a trace from the null control line 15 (red). Detector response is measured in nC, retention time in minutes. G3 and G4 are glucan fragments released from β-glucan by digestion with lichenase; X and XX are Xyl and xylobiose released from AX by digestion with endoxylanase. Peaks 1 to 7 are AXOS released from AX by digestion with endoxylanase: 1 = XA³X, 2 = XA³XX, 3 = XA²⁺³XX, 4 = XA³A³XX, 5 = XA³XA³XX, 6 = XA³A²⁺³XX, and 7 = XA³XA²⁺³XX. All identified peaks were integrated (Supplemental Table S4) and used for PCA analysis (Fig. 4; Supplemental Fig. S5).

Figure 4.

PCA of the enzyme fingerprinting (HPAEC) results of mature seeds of five transgenic and three control lines. The loading plot (see Supplemental Fig. S5) shows that the first principal component PC1 is mainly related to variation in G3 and G4 peaks and thus indicated variation in the proportions of β-glucans among samples. Samples on the right part of the plot are depleted in β-glucans compared to samples on the left part of the plot.

Figure 5.

Areas of HPAEC peaks corresponding to G3 + G4 fragments in mature grain (A) and developing caryopses (B) of transgenic (4T, 7T, 9T, 15T, and 21T) and control (15N, 20N, and 23C) lines. Values in bars show G3 to G4 ratios in the different lines. daa, Days after anthesis. [See online article for color version of this figure.]

Figure 6.

se-HPLC profiles showing the M_r distribution of water-extractable β-glucan from wholemeal samples of mature grain of transgenic (9T, 21T, and 15T) and control (20N, 23C, and 15N) lines of wheat. The dotted lines represent the Calcofluor average M_rs.

Figure 7.

Immunolabeling of β-glucan in mature grain (A–D) and developing caryopses (E–J) of transgenic (sections on left) and control (sections on right) lines of wheat. Bars = 100 μm. al, Aleurone layer; en, starchy endosperm; ne, nucellar epidermis; pe, pericarp.