Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

Abstract

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed.

Fig. 1.

Diagrammatic representation of the RNA silencing constructs (not drawn to scale). (A) A 21 nucleotide artificial microRNA (ami-BEIIb)-based osa-miR528 was synthesized by fusion PCR and cloned into Vec8, a Ti binary vector with a wheat high molecular weight glutenin promoter (wHMGPro) and nopaline synthase terminator (NOS). (B) The secondary structure of the osa-miR528 backbone as predicted by RNAfold, including information on ami-BEIIb (reverse complement). The predicted target cleavage site (arrow with sequence bold and highlighted) is located between positions 10 and 11 of the amiRNA, while the two mismatches (grey highlight) are located at positions 1 and 21. (C) The hairpin RNA (hp-BEIIb) was cloned in Vec8 by inserting a 397 bp BEIIb fragment in the sense (BEIIb→) and antisense (BEIIb←) orientations. The two fragments are flanked by two rice introns, Rint4 and Rint9, which form a hairpin loop. The amiRNA and hp-RNA fragments were directionally cloned using several restriction sites (H, HindIII; B, BamHI; K, KpnI; N, NotI; E EcoRI; S, SpeI).

Fig. 2.

Gene expression analyses by quantitative RT-PCR. The expression of SBEIIb was reduced 5-fold in ami-BEIIb lines and only 2-fold in hp-BEIIb lines. The expression of other branching enzymes and major starch synthases was unaffected. The rice tubulin gene was used as a reference (Toyota et al., 2006) for comparative quantitation. Mean values with different letters are significantly different. Error bars indicate the SEM.

Fig. 3.

Enzyme expression and activity detection using western blots (A and B) and a zymogram (C) using 10 dpa grains. The levels of BEIIb (A) in rice endosperm are down-regulated to undetectable levels in ami-BEIIb (A1–A3) and to trace amounts in hp-BEIIb (R1–R3) lines. The levels of BEIIa (B) are similar for the transgenic and parental Nipponbare (NB) lines. Enzyme activity detection (C) shows that BEIIb activity is only detectable in NB but not in the transgenic lines, while BEIIa activity remains intact. This result is corroborated by reducing end assay (D) which shows a decreased concentration of reducing ends in the transgenic lines which is more pronounced in ami-BEIIb. Mean values with different letters are significantly different. Error bars indicate the SEM.

Fig. 4.

Grain and starch granule morphology of transgenic lines. The polished grain of ami-BEIIb (A) appeared chalky, and that of hp-BEIIb (B) had some chalky character, compared with the translucent grains of Nipponbare tissue culture control (C). At ×1000 magnification, the starch granules of the transformed lines are loose and rounded (D and E), compared with the tight and angular granules in the control (F). Big and small rounded starch granules were observed in the transformed lines. The difference in starch granule morphology appears to be more pronounced in the ami-BEIIb lines (D). Actual grain dimensions are reported in Table 1.

Fig. 5.

XRD patterns of starches with down-regulated SBEIIb and its comparison with the wild type and the amylose extender mutant. Two hp-BEIIb lines (hp-BEIIb A-type) share the same A-type crystalline polymorph (peak at 18 °, but not 5 °, 2θ) with the wild-type rices (Nipponbare and IR36) while all four ami-BEIIb shifted to a B-type polymorph (peak at 5 °, but not 18 °, 2θ) similar to IR36ae. Two hp-BEIIb showed a crystalline structure that is intermediate between A- and B-type (hp-BEIIb C-type). Data are off set for clarity.

Fig. 6.

Chain length distribution (CLD) profile of debranched starch (A and B) and mol% difference of ami-BEIIb and hp-BEIIb compared with Nipponbare (C). Compared with Nipponbare, ami-BEIIb (A) and hp-BEIIb (B) have reductions in DP 6–12 and an increase in DP 13 onwards. A difference plot (C) revealed that the reduction in short DP is more pronounced in ami-BEIIb (up to 4%) than in hp-BEIIb (up to 3%). Likewise, the increase in longer DPs is more pronounced in ami-BEIIb (up to 0.9%) than in hp-BEIIb (up to 0.7%). Error bars indicate the SEM.

Fig. 7.

Debranched HP-SEC of transformed lines compared with Nipponbare using (A) Ultrahydrogel 250 and (B) Proteema columns. Intermediate length chains (apparent DP ≥37) were increased while the short amylopectin chains (DP ≤36) were decreased in both hp-SBEIIb and ami-BEIIb, with the changes more pronounced in the latter. No change in the proportion of long chain amylose peak (apparent DP ≥1000) was observed, while the mutants have elevated amounts of intermediate material—longer than traditionally found in amylopectin (DP 6–120) and shorter than classic long chain amylose (inset).