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 β-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

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

Abstract 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.</p

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

Accumulation of storage products in the developing barley caryopsis.

<p>(a) Starch, (b) protein and (c) BG, deposition of starch (d) amylose and (e) phosphate in WT, HP and AO at 10, 20, 30 DAP and Mature dry grain developmental stages. Values are means of five biological replicates ± SE. Asterisks indicate significant differences by one-way anova of transgenic lines compared with the WT at the same developmental stage (* P<0.01, ** P<0.001). For clarity, difference in P value is not indicated where obvious difference is observed. HP: ◯ WT: ● AO:▼.</p

Relative levels of primary metabolites in the developing barley caryopsis of WT (red), HP (green) and AO (blue) at 10, 20, 30 DAP and MDG.

<p>The data is normalized to the WT 10 DAP sample and log2 transformed and presented as boxplots. The box and the horizontal line within the box represent the data at first and third quartile and median, respectively. The ends of vertical bars show maximum and minimum values. Student's <i>t</i>-test was performed to evaluate statistical difference between WT and transgenic plants for each time point by <i>t</i>-test. * indicates significant difference (P<0.05) to WT at each time point.</p

Principal component analysis of the metabolite profiles of grains during grain development.

<p>Each symbol represents the mean of all biological replicates (n = 6). The shape of symbols represents the time points. Cross, mature grain; circle, 10 days after pollination (DAP); triangle, 20 DAP; square, 30 DAP. The colours of the symbols are corresponding to the genotype. Red, AO line; green, HP line; blue, wild type.</p

Single starch granule topography and morphology of selected typical specimens analysed by non-coated scanning electron microscopy.

<p>Starch purified from developing caryopsis of WT (a-d), HP (e-h) and AO (i-l) at 10, 20, 30 DAP and mature dry grain developmental stages. Red arrows indicate rare surface pores in HP. Scale bar indicates 20 μm.</p

Mature dry grain (MDG) starch, amylose, starch bound C6 phosphate, BG and protein contents of WT and the transgenic lines.

<p>Starch, BG and protein contents were determined on the mature dry grains. Amylose and starch bound C6 phosphate was determined on purified starch. Values are presented as means ± SE of five biological replicates.</p