Non-GMO potato lines, synthesizing increased amylose and resistant starch, are mainly deficient in isoamylase debranching enzyme.

Solanum tuberosum potato lines with high amylose content were generated by crossing with the wild potato species Solanum sandemanii followed by repeated backcrossing to Solanum tuberosum lines. The trait, termed increased amylose (IAm), was recessive and present after three generations of backcrossing into S. tuberosum lines (6.25% S. sandemanii genes). The tubers of these lines were small, elongated and irregular with small and misshaped starch granules and high sugar content. Additional backcrossing resulted in less irregular tuber morphology, increased starch content (4.3%-9.5%) and increased amylose content (29%-37.9%) but indifferent sugar content. The amylose in the IAm starch granules was mainly located in peripheral spots, and large cavities were found in the granules. Starch pasting was suppressed, and the digestion-resistant starch (RS) content was increased. Comprehensive microarray polymer profiling (CoMPP) analysis revealed specific alterations of major pectic and glycoprotein cell wall components. This complex phenotype led us to search for candidate IAm genes exploiting its recessive trait. Hence, we sequenced genomic DNA of a pool of IAm lines, identified SNPs genome wide against the draft genome sequence of potato and searched for regions of decreased heterozygosity. Three regions, located on chromosomes 3, 7 and 10, respectively, displayed markedly less heterozygosity than average. The only credible starch metabolism-related gene found in these regions encoded the isoamylase-type debranching enzyme Stisa1. Decreased expression of mRNA (>500 fold) and reduced enzyme activity (virtually absent from IAm lines) supported Stisa1 as a candidate gene for IAm.

Starch Granule Re-Structuring by Starch Branching Enzyme and Glucan Water Dikinase Modulation Affects Caryopsis Physiology and Metabolism.

Starch is of fundamental importance for plant development and reproduction and its optimized molecular assembly is potentially necessary for correct starch metabolism. Re-structuring of starch granules in-planta can therefore potentially affect plant metabolism. Modulation of granule micro-structure was achieved by decreasing starch branching and increasing starch-bound phosphate content in the barley caryopsis starch by RNAi suppression of all three Starch Branching Enzyme (SBE) isoforms or overexpression of potato Glucan Water Dikinase (GWD). The resulting lines displayed Amylose-Only (AO) and Hyper-Phosphorylated (HP) starch chemotypes, respectively. We studied the influence of these alterations on primary metabolism, grain composition, starch structural features and starch granule morphology over caryopsis development at 10, 20 and 30 days after pollination (DAP) and at grain maturity. While HP showed relatively little effect, AO showed significant reduction in starch accumulation with re-direction to protein and ß-glucan (BG) accumulation. Metabolite profiling indicated significantly higher sugar accumulation in AO, with re-partitioning of carbon to accumulate amino acids, and interestingly it also had high levels of some important stress-related metabolites and potentially protective metabolites, possibly to elude deleterious effects. Investigations on starch molecular structure revealed significant increase in starch phosphate and amylose content in HP and AO respectively with obvious differences in starch granule morphology at maturity. The results demonstrate that decreasing the storage starch branching resulted in metabolic adjustments and re-directions, tuning to evade deleterious effects on caryopsis physiology and plant performance while only little effect was evident by increasing starch-bound phosphate as a result of overexpressing GWD.

Starch bioengineering affects cereal grain germination and seedling establishment.

Cereal grain germination is central for plant early development, and efficient germination has a major role in crop propagation and malting. Endosperm starch is the prime energy reserve in germination and seedling establishment. In this study, it was hypothesized that optimized starch granule structure, and not only the endosperm starch content per se, is important for germination and seedling establishment. For that purpose, wild-type (WT), and specifically engineered degradable hyperphosphorylated (HP) starch and more resistant amylose-only (AO) starch barley lines were used. The transgenics showed no severe phenotypes and the WT and HP lines degraded the starch similarly, having 30% residual starch after 12 d of germination. However, the AO line showed significant resistance to degradation, having 57% residual starch. Interestingly, protein and ß-glucan (BG) degradation was stimulated for both HP and AO lines as compared with the WT. At late seedling establishment stages, specific sugars were rapidly consumed in the AO line. α-Amylase activity was distinctly suppressed in both the HP and the AO lines. Pre-germination ß-amylase deposition was low in the AO grains and ß-amylase was generally suppressed in both HP and AO lines throughout germination. As further supported by scanning electron microscopy and histochemical analyses on grain and seedlings, it was concluded that inadequate starch granule deposition in combination with the suppressed hydrolase activity leads to temporal and compensating re-direction of starch, sugar, and protein catabolism important to maintain metabolic dynamics during grain germination and seedling establishment.

Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules.

BACKGROUND: Starch is stored in higher plants as granules composed of semi-crystalline amylopectin and amorphous amylose. Starch granules provide energy for the plant during dark periods and for germination of seeds and tubers. Dietary starch is also a highly glycemic carbohydrate being degraded to glucose and rapidly absorbed in the small intestine. But a portion of dietary starch, termed "resistant starch" (RS) escapes digestion and reaches the large intestine, where it is fermented by colonic bacteria producing short chain fatty acids (SCFA) which are linked to several health benefits. The RS is preferentially derived from amylose, which can be increased by suppressing amylopectin synthesis by silencing of starch branching enzymes (SBEs). However all the previous works attempting the production of high RS crops resulted in only partly increased amylose-content and/or significant yield loss. RESULTS: In this study we invented a new method for silencing of multiple genes. Using a chimeric RNAi hairpin we simultaneously suppressed all genes coding for starch branching enzymes (SBE I, SBE IIa, SBE IIb) in barley (Hordeum vulgare L.), resulting in production of amylose-only starch granules in the endosperm. This trait was segregating 3:1. Amylose-only starch granules were irregularly shaped and showed peculiar thermal properties and crystallinity. Transgenic lines retained high-yield possibly due to a pleiotropic upregualtion of other starch biosynthetic genes compensating the SBEs loss. For gelatinized starch, a very high content of RS (65 %) was observed, which is 2.2-fold higher than control (29%). The amylose-only grains germinated with same frequency as control grains. However, initial growth was delayed in young plants. CONCLUSIONS: This is the first time that pure amylose has been generated with high yield in a living organism. This was achieved by a new method of simultaneous suppression of the entire complement of genes encoding starch branching enzymes. We demonstrate that amylopectin is not essential for starch granule crystallinity and integrity. However the slower initial growth of shoots from amylose-only grains may be due to an important physiological role played by amylopectin ordered crystallinity for rapid starch remobilization explaining the broad conservation in the plant kingdom of the amylopectin structure.