Production of very-high-amylose cassava by post-transcriptional silencing of branching enzyme genes

Abstract

High amylose starch, a desired raw material in the starch industry, can be produced by plants deficient in the function of branching enzymes (BEs). Here we report the production of transgenic cassava plants with starches containing up to 50% amylose due to the constitutive expression of hair-pin dsRNAs targeting the BE1 or BE2 genes. A significant decrease in BE transcripts was confirmed in these transgenic plants by quantitative real-time RT-PCR. The absence of BE1 protein in the BE1-RNAi plant lines (BE1i) and a dramatically lower level of BE2 protein in the BE2-RNAi plant lines (BE2i) were further confirmed by Western blot assays. All transgenic plant lines were grown up in the field, but with reduced biomass production of the above-ground parts and storage roots compared to wild type (WT). Considerably high amylose content in the storage roots of BE2i plant lines was achieved, though not in BE1i plant lines. Storage starch granules of BE1i and BE2i plants had similar morphology as WT, however, the size of BE1i starch granules were bigger than that of WT. Comparisons of amylograms and thermograms of all three sources of storage starches revealed dramatic changes to the pasting properties and a higher melting temperature for BE2i starches. Glucan chain length distribution analysis showed a slight increase in chains of DP>36 in BE1i lines and a dramatic increase in glucan chains between DP 10-20 and DP>40 in BE2i lines, compared to that of WT starch. Furthermore, BE2i starches displayed a B-type X-ray diffraction pattern instead of the A-type pattern found in BE1i and WT starches. Therefore, cassava BE1 and BE2 function differently in storage root starch biosynthesis; silencing of cassava BE1 or BE2 caused various changes to starch physico-chemical properties and amylopectin structure. We also report that remarkably high amylose content in cassava starch has been first obtained in transgenic cassava by silencing of BE2 expression, thus showing a high potential for future industrial utilization.