Control of starch branching in barley defined through differential RNAi suppression of starch branching enzyme IIa and IIb

Abstract

The roles of starch branching enzyme (SBE, EC 2.4.1.18) IIa and SBE IIb in defining the structure of amylose and amylopectin in barley (Hordeum vulgare) endosperm were examined. Barley lines with low expression of SBE IIa or SBE IIb, and with the low expression of both isoforms were generated through RNA-mediated silencing technology. These lines enabled the study of the role of each of these isoforms in determining the amylose content, the distribution of chain lengths, and the frequency of branching in both amylose and amylopectin. In lines where both SBE IIa and SBE IIb expression were reduced by >80%, a high amylose phenotype (>70%) was observed, while a reduction in the expression of either of these isoforms alone had minor impact on amylose content. The structure and properties of the high amylose starch resulting from the concomitant reduction in the expression of both isoforms of SBE II in barley were found to approximate changes seen in amylose extender mutants of maize, which result from lesions eliminating expression of the SBE IIb gene. Amylopectin chain length distribution analysis indicated that both SBE IIa and SBE IIb isoforms play distinct roles in determining the fine structure of amylopectin. A significant reduction in the frequency of branches in amylopectin was noticed only when both SBE IIa and SBE IIb were reduced, whereas there was a significant increase in the branching frequency of amylose when SBE IIb alone was reduced. Functional interactions between SBE isoforms are suggested, and a possible inhibitory role of SBE IIb on other SBE isoforms is discussed.

Fig. 1.

(A, B) Southern hybridization of genomic DNA from barley transgenic lines. ApaI digested genomic DNA extracted from individual plants of transgenic barley lines was hybridized to a probe from the intron 3 region of SBE IIa (A) and SBE IIb (B) genes. (A) Lanes 1 and 2 are two different T₂ lines of the same transformation event, IIa 4 and IIa 4.1, lanes 3 and 7 are non transformed control (WT), lanes 4–6 are F₂ barley lines of the cross between IIa 4×IIb 4, BC 1, BC 2, and BC 3, and lane 8 is a T₃ line of IIa 4. (B) Lane 1 is a T₂ line, IIb 4, lanes 2 and 6 are WT, lane 3–5 are same F₂ lines as the lanes 4–6 of (A), and lane 7 is a T₃ line of IIb 4.

Fig. 2.

Immunodetection of SBE IIa and SBE IIb in developing endosperms of barley transgenic lines. (A, B) Segregation of SBE IIa (A) and SBE IIb (B) expression in seven T₁ developing endosperms (lanes 2–8) from the T₀ barley hp-SBE IIa transgenic line IIa 4 as shown by immunoblotting using anti-SBE IIa and anti-SBE IIb antibodies, respectively. Lane 1 is WT. (C, D) Segregation of SBE IIb (C) and SBE IIa (D) expression in six T₁ developing endosperms (lanes 1–6) from the T₀ barley hp-SBE IIb transgenic line IIb 4 as shown by immunoblotting using anti-SBE IIb and anti-SBE IIa antibodies, respectively. Lane 7 is WT. (E) The relative levels of expression of SBE IIa and SBE IIb in selected barley transgenic lines, SBE IIa^–, SBE IIb^– , SBE IIa^–/SBE IIb↓, and SBE IIa^–/SBE IIb^–. Immunoblots of non-denaturing PAGE scanned and the band intensities of the images measured using the TotalLab software package (Nonlinear Dynamics Ltd, Newcastle, UK). The expression of SBE IIa and SBE IIb in each transgenic line is expressed as a percentage of the level of expression of the respective isoform in WT.

Fig. 3.

Comparison of branching enzyme in barley transgenic lines. Electrophoresis of soluble proteins (15 μg well⁻¹) from 15 dpa endosperms was performed as described in the Materials and methods. (A) Activity staining of branching enzymes. After electrophoresis the gel was washed twice and incubated with maltotriose and visualized with iodine. (B, C) Immunoblots. The membranes were probed with SBE IIa antibody (B) and SBE IIb antibody (C). The samples were: lane 1, WT; lane 2, SBE IIa^–; lane 3, SBE IIb^–; lane 4, SBE IIa^–/SBE IIb^–.

Fig. 4.

Transcript expression in transgenic barley lines. Expression of SBE IIa (A) and SBE IIb (B) transcripts in transgenic barley lines compared with the non-transformed control (WT) determined by comparative quantitation using real-time PCR. The expression of the SBE II isoform is normalized to expression of the house-keeping gene actin from respective lines. The average of two biological replicates is presented in the figure.

Fig. 5.

Scanning electron micrographs of isolated starch granules (1000× magnification). (A) WT, (B) SBE IIb^–, (C) SBE IIa^–, (D) SBE IIa^–/SBE IIb↓, and (E) SBE IIa^–/SBE IIb^– lines.

Fig. 6.

Sepharose CL 2B gel chromatogram of starch from barley transgenic lines. (A) WT, (B) SBE IIb^–, (C) SBE IIa^–, (D) SBE IIa^–/SBE IIb↓, and (E) SBE IIa^–/SBE IIb^–. Starch molecules separated by gel filtration are assayed using starch assay kit (Sigma). Amylose content estimated by this method as a percentage of total starch is shown on respective graphs.

Fig. 7.

Chain length profile comparison of starches from barley transgenic lines with respect to that of WT. The percentage of total mass of the individual oligosaccharides from starches from respective non-transformed controls is subtracted from the corresponding values from starches from transgenic lines. Samples are SBE IIa^– (filled triangles), SBE IIa^–/SBE IIb^– (open diamonds), SBE IIb^– (filled squares).

Fig. 8.

RVA and DSC studies on starches from barley transgenic lines. (A) RVA profile of starches from WT, SBE IIa^–, SBE IIb^–, and SBE IIa^–/SBE IIb^–. (B) DSC profile of starches from WT, SBE IIa^–, SBE IIb^–, and SBE IIa^–/SBE IIb^–.