Combining mutations at genes encoding key enzymes involved in starch synthesis affects the amylose content, carbohydrate allocation and hardness in the wheat grain

Abstract

Modifications to the composition of starch, the major component of wheat flour, can have a profound effect on the nutritional and technological characteristics of the flour's end products. The starch synthesized in the grain of conventional wheats (Triticum aestivum) is a 3:1 mixture of the two polysaccharides amylopectin and amylose. Altering the activity of certain key starch synthesis enzymes (GBSSI, SSIIa and SBEIIa) has succeeded in generating starches containing a different polysaccharide ratio. Here, mutagenesis, followed by a conventional marker-assisted breeding exercise, has been used to generate three mutant lines that produce starch with an amylose contents of 0%, 46% and 79%. The direct and pleiotropic effects of the multiple mutation lines were identified at both the biochemical and molecular levels. Both the structure and composition of the starch were materially altered, changes which affected the functionality of the starch. An analysis of sugar and nonstarch polysaccharide content in the endosperm suggested an impact of the mutations on the carbon allocation process, suggesting the existence of cross-talk between the starch and carbohydrate synthesis pathways.

Keywords: amylose; carbohydrates allocation; cell wall polysaccharides; gene expression; kernel hardness; resistant starch.

Figure 1

Crystallinity and viscosity in the mutant wheats and the control Cadenza. (a) X‐ray diffraction patterns of starch synthesized by (A) cv Cadenza (B) Cad‐GBSSI* (C) Cad‐SSIIa* and (D) Cad‐SBEIIa*. Peaks diagnostic for A‐ and V‐type crystals are indicated. The intensity is given in arbitrary units (a. u.). The asterisks indicate the presence of impurities in the sample. (b) Viscosity profiles as measured by RVA (A) cv Cadenza, (B) Cad‐GBSSI*, (C) Cad‐SSIIa* and (D) Cad‐SBEIIa*.

Figure 2

Starch granule morphology and amylopectin branched chains length in the set of mutant genotypes and the control. (a) Scanning electron micrographs of starch granules in flour prepared from (A) cv. Cadenza, (B) Cad‐GBSSI*, (C) Cad‐SSIIa* and (D) Cad‐SBEIIa*. (b) (A) Chain length distribution of amylopectin in the control cv Cadenza; the histograms illustrate the differences in chain length distribution between cv. Cadenza and the mutant lines and: (B) Cad‐GBSSI*, (C) Cad‐SSIIa* and (D) Cad‐SBEIIa*.

Figure 3

Total soluble carbohydrate content in the flour of cv. Cadenza and its derived null mutant lines: (A) cv. Cadenza, (B) Cad‐GBSSI*, (C) Cad‐SSIIa* and (D) Cad‐SBEIIa*. The full bar represents the total soluble carbohydrate content expressed as mg/g. The composition of the soluble carbohydrates is reported as the sum of glucose, fructose, 1‐kestose, sucrose, raffinose, maltose, galactose, water‐soluble α‐glucans and fructans.

Figure 4

The transcription of genes encoding key enzymes in carbon allocation in the endosperm as estimated by qRT‐PCR. Cad‐SBEIIa* is shown by black bars, Cad‐SSIIa* by grey bars and Cad‐GBSSI* by white bars. Genes involved in (a) sugar cleavage and cytosolic synthesis of ADP‐glucose, (b) 6&1 fructan exohydrolase (6&1FEH), (c) starch synthesis in the amyloplast, (d) arabinoxylan and β‐glucan synthesis. Each bar represents the mean of three biological replicates and standard errors are indicated by thin black bars. The level of expression of the control is 1.

Figure 5

A schematic illustration of metabolic flux and of the transcripts level of the genes involved in the pathways of sucrose, starch, β‐glucans, arabinoxylans and fructans in the endosperm of the three mutant lines: panel (a) Cad‐GBSSI*; panel (b) Cad‐SSIIa*; and panel (c) Cad‐SBEIIa*. Squares indicate metabolites; circles stand for genes. Dashed arrows indicate the overall flux whereas the solid arrows stand for the single reaction. Full colours indicate, for genes, increase in expression of 3‐fold or more (blue), and decrease of expression of 3‐fold or less (red); for metabolites, statistically significant changes of +100% or more (blue), or −50% or less (red). Thresholds for intermediate colours were: increase in gene expression (between 2‐ and 3‐fold; light red), decrease in gene expression (between 2‐ and 3‐fold); metabolites: light blue when the increase was significant but less than 100%; light red when significant but less than −50%.