Increasing the amylose content of durum wheat through silencing of the SBEIIagenes

Abstract

BackgroundHigh amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter.ResultsAmylose content was markedly increased in the durum wheat transgenic lines exhibiting SBEIIa gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR).ConclusionWe have found that the silencing of SBEIIa genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable.

Creation of a high-amylose durum wheat through mutagenesis of starch synthase II (SSIIa)

“…Wx-A1, SsI-1, SbeI-A, SbeIIa-A, SbeIIa-B, SSIII, AgpL (large subunit of AGPase), and Pho1 (alpha-1,4-glucan phosphorylase) transcript levels were increased in the SGP-1 double null lines. The up-regulation of starch biosynthetic genes after the elimination of a key enzyme has also been observed in bread wheat where SbeIIa was silenced using RNAi (Sestili et al, 2010b). Using qRT-PCT, Sestili et al (2010b) observed increases in Wx-1, SSIII, Iso1, and Ld1 transcripts but no increase for SsI, SSIIa, SbeIIb, or SbeI.…”

“…durum) cultivar 'Svevo' and hexaploid SGP-A1/B1 null lines (Yamamori and Endo, 1996) had an 89% increase in amylose content compared to Svevo (43.6% SGP-1 null vs 23% wild-type) as well as reduced binding of SGP-2 and SGP-3. Elimination of SbeIIa in durum through RNA interference also resulted in increased amylose ranging from þ29% to þ200% (24% wild-type vs. 31e75% SbeIIa RNAi lines) (Sestili et al, 2010b). The very high amylose results observed by Sestili et al (2010b) may not be due solely to SbeIIa expression reduction since SbeIIa mutants have amylose level increases similar to those of SSIIa mutations (28% sbeIIa versus 23% wild-type) (Hazard et al, 2012).…”

“…Elimination of SbeIIa in durum through RNA interference also resulted in increased amylose ranging from þ29% to þ200% (24% wild-type vs. 31e75% SbeIIa RNAi lines) (Sestili et al, 2010b). The very high amylose results observed by Sestili et al (2010b) may not be due solely to SbeIIa expression reduction since SbeIIa mutants have amylose level increases similar to those of SSIIa mutations (28% sbeIIa versus 23% wild-type) (Hazard et al, 2012).…”

Biotechnology for Enhanced Nutritional Quality in Plants

“…Transgenic wheat expressing the double strand RNA targeting SBEII-b did not display a significant increase in starch amylose content while RNAi silencing of SBEII-a resulted in an endosperm starch content with more than 70% amylose. Sestili et al (2010) followed the same strategy with two cultivars of tetraploid durum wheat (T. turgidum L. var. durum).…”

Plant Biotechnology for the Development of Design Starches

“…The alteration of starch quantity and quality can be achieved through the overexpression of some enzymes involved in starch synthesis [60], by mutations or RNAi technology, such as the inhibition of potato SSII, SSIII and GBSS [61], or the decrease in the expression of wheat BEIIa and BEIIb [62,63]. In this way, by affecting the catalytic activity of enzymes involved in the synthesis of amylose or amylopectin, it could be possible to obtain starches for different purposes.…”