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Outlines the key parameters which should be considered when attempting the study of plant membrane proteins in artificial lipid bilayers. Solubilization of the protein with a suitable surfactant; Functional reconstitution of the protein in a well characterized liposome system.

This study investigated the influence of diurnal photosynthetic activity on the morphology, molecular composition, crystallinity, and gelatinization properties of normal barley starch (NBS) and waxy barley starch (WBS) granules from plants cultivated in a greenhouse under normal diurnal (16h light) or constant light photosynthetic conditions. Growth rings were observed in all starch samples regardless of lighting conditions. The size distribution of whole and debranched WBS analyzed by gel-permeation chromatography did not appear to be influenced by the different lighting regimes, however, a greater relative crystallinity measured by wide-angle X-ray scattering and greater crystalline quality as judged by differential scanning calorimetry was observed under the diurnal lighting regime. NBS cultivated under the diurnal photosynthetic lighting regime displayed lower amylose content (18.7%), and shorter amylose chains than its counterpart grown under constant light. Although the relative crystallinity of NBS was not influenced by lighting conditions, lower onset, peak, and completion gelatinization temperatures were observed in diurnally grown NBS compared to constant light conditions. It is concluded that normal barley starch is less influenced by the diurnal photosynthetic lighting regime than amylose-free barley starch suggesting a role of amylose to prevent structural disorder and increase starch granule robustness against environmental cues., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The amylopectin fractions from starch of a series of amylose-extender (ae) maize samples (HYLON(®) V, VII and VIII starches) were isolated and analysed for their molecular composition and structure. The fractions from all samples contained both a high and a low molecular weight fraction (HMF and LMF), of which LMF increased with the amylose content of the starch and appeared to have substantially more of long chains than HMF. A normal amylose-containing maize starch (NMS), which served as a reference sample, contained very little LMF, which suggested that LMF was the inherent result of the effect of the loss of starch branching enzyme IIb activity in the ae mutants. Clusters were isolated from the amylopectin fractions using Bacillus amyloliquefaciens α-amylase, which effectively hydrolyses long internal chain segments between clusters. During the hydrolysis process, clearly more of small dextrins were released from the ae starches in comparison to NMS. It appeared that some of these small dextrins did not precipitate in methanol together with the majority of the clusters. Nevertheless, isolated clusters from the HYLON starch samples were smaller than in NMS and the clusters possessed a lower density of branches with longer chains. The composition of small, branched building blocks was also clearly different: HYLON starch samples possessed much more of single-branched blocks and less multiple-branched blocks than NMS., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Little is known about the thermal properties and internal molecular structure of transitory starch. In this study, granule morphology, thermal properties, and the cluster structure of Arabidopsis leaf starch at beginning and end of the light period were explored. The structural properties of building blocks and clusters were evaluated by using diverse chromatographic techniques. On the granular level, starch from end of day had larger granule size, thinner crystalline lamellae thickness, lower free surface energy of crystals, and lower tendency to retrograde than that from end of night. On the molecular level, the starch had lower amylose content, larger cluster size, and higher number of blocks per cluster at the end of day than at end of night. It is concluded that the core of the granules contains a more permanent molecular and less-ordered physical structure different from the transitory layers laid down around the core at daytime., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In this study, starches extracted from wheat grains harvested at 7, 14, 28, and 35 days after anthesis (DAA) were used as a means of examining the molecular structure of amylopectin (AP) from developing wheat grain. Scanning electron microscopy of wheat grain cross-sections revealed the presence of endosperm at 7 DAA and contained lenticular-shaped developing large (A-type) granules. From 14 DAA onward, spherical-shaped small (B-type) granules coexisted with large granules in the endosperm. During granule development, the fine structure of AP varied with maturity in both large and small granules. Towards the end of the pre-physiological maturity stage (28 DAA), AP in small and large granules had shortest external chain length (ECL), longest internal chain length (ICL) and lowest amount of A-chains. At physiological maturity (35 DAA), these changes in ECL, ICL and amount of A-chains were reversed when compared to 28 DAA. In both large and small granules, the external AP structure was apparently more organized at physiological maturity than at pre-physiological maturity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Changes in internal structure of amylopectin (AP) during wheat endosperm development were studied by isolating clusters and building blocks of AP from both large A-type and small B-type starch granules at different maturity stages up to harvest time at 49 days after anthesis (DAA). Clusters isolated from B-type granules had a degree of branching (DB) of 16.5-16.8% and were more tightly branched than those isolated from A-type granules (DB 15.7-16.2%). The degree of polymerization (DP) of the clusters increased in both types of granules during the pre-physiological maturity stage up to 28 DAA. Clusters at maturity were smaller with less branches and building blocks than at the end of the pre-maturity stage. It is suggested that this was due to a continuous trimming of the cluster structure after the active period of starch synthesis. Differences were evident between A- and B-type granules with regards to glucan trimming and the type of new chains produced., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Elevated proportions of amylose in cereals are commonly associated with either the loss of starch branching or starch synthase activity. Goami 2 is a high-amylose mutant of the temperate japonica rice variety Ilpumbyeo. Genotyping revealed that Goami 2 and Ilpumbyeo carry the same alleles for starch synthase IIa and granule-bound starch synthase I genes. Analyses of granule-bound proteins revealed that SSI and SSIIa accumulate inside the mature starch granules of Goami 2, which is similar to the amylose extender mutant IR36ae. However, unlike the amylose extender mutants, SBEIIb was still detectable inside the starch granules of Goami 2. Detection of SBEIIb after protein fractionation revealed that most of the SBEIIb in Goami 2 accumulates inside the starch granules, whereas most of it accumulates at the granule surface in Ilpumbyeo. Exhaustive mass spectrometric characterisations of granule-bound proteins failed to detect any peptide sequence mutation or major post-translational modifications in Goami 2. Moreover, the signal peptide was found to be cleaved normally from the precursor protein, and there is no apparent N-linked glycosylation. Finally, no difference was found in the SBEIIb structural gene sequence of Goami 2 compared with Ilpumbyeo. In contrast, a G-to-A mutation was detected in the SBEIIb gene of IR36ae located at the splice site between exon and intron 11, which could potentially introduce a premature stop codon and produce a truncated form of SBEIIb. It is suggested that the mutation responsible for producing high amylose in Goami 2 is not due to a defect in SBEIIb gene as was observed in IR36ae, even though it produces a phenotype analogous to the amylose extender mutation. Understanding the molecular genetic basis of this mutation will be important in identifying novel targets for increasing amylose and resistant starch contents in rice and other cereals.

Amyloplasts were isolated and purified from wheat endosperm and the envelope membranes reconstituted into liposomes. Envelope membranes were solubilized in n-octyl beta-D-glucopyranoside and mixed with liposomes supplemented with 5.6 mol% cholesterol to produce proteoliposomes of defined size, which showed negligible leakage of internal substrates. Transport experiments with proteoliposomes revealed a counter-exchange of glucose 1-phosphate (Glc1P), glucose 6-phosphate (Glc6P), inorganic phosphate (Pi), 3-phosphoglycerate and dihydroxyacetone phosphate. The Glc1P/Pi counter-exchange reaction exhibited an apparent K(m) for Glc1P of 0.4 mM. Glc6P was a competitive inhibitor of Glc1P transport (Ki 0.8 mM), and the two hexose phosphates could exchange with each other, indicating the operation of a single carrier protein. Glc1P/Pi antiport in proteoliposomes showed an exchange stoichiometry at pH 8.0 of 1 mol of phosphate transported per mol of sugar phosphate.