CRISPR-Cas9-mediated editing of starch branching enzymes results in altered starch structure in Brassica napus.

Starch branching enzymes (SBEs) are one of the major classes of enzymes that catalyze starch biosynthesis in plants. Here, we utilized the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9)-mediated gene editing system to investigate the effects of SBE mutation on starch structure and turnover in the oilseed crop Brassica napus. Multiple single-guide RNA (sgRNA) expression cassettes were assembled into a binary vector and two rounds of transformation were employed to edit all six BnaSBE genes.

Protein phosphorylation regulates maize endosperm starch synthase IIa activity and protein-protein interactions.

Starch synthesis is an elaborate process employing several isoforms of starch synthases (SSs), starch branching enzymes (SBEs) and debranching enzymes (DBEs). In cereals, some starch biosynthetic enzymes can form heteromeric complexes whose assembly is controlled by protein phosphorylation. Previous studies suggested that SSIIa forms a trimeric complex with SBEIIb, SSI, in which SBEIIb is phosphorylated.

AKINβ1, a subunit of SnRK1, regulates organic acid metabolism and acts as a global modulator of genes involved in carbon, lipid, and nitrogen metabolism.

The sucrose non-fermenting-1-related protein kinase 1 (SnRK1) is a highly conserved heterotrimeric protein kinase in plants. It possesses a catalytic subunit (α) and two regulatory subunits (β and γ). The effects of altered expression of AKINβ1 on carbohydrate metabolism and gene expression in leaves were investigated in an Arabidopsis T-DNA insertion mutant.

Bioinformatic and Analyses of Arabidopsis Starch Synthase 2 Reveal Post-translational Regulatory Mechanisms.

Starch synthase 2 (SS2) is an important enzyme in leaf starch synthesis, elongating intermediate-length glucan chains. Loss of SS2 results in a distorted starch granule phenotype and altered physiochemical properties, highlighting its importance in starch biosynthesis, however, the post-translational regulation of SS2 is poorly understood. In this study, a combination of bioinformatic and analysis of recombinant SS2 was used to identify and characterize SS2 post-translational regulatory mechanisms.

Starch as a source, starch as a sink: the bifunctional role of starch in carbon allocation.

Starch commands a central role in the carbon budget of the majority of plants on earth, and its biological role changes during development and in response to the environment. Throughout the life of a plant, starch plays a dual role in carbon allocation, acting as both a source, releasing carbon reserves in leaves for growth and development, and as a sink, either as a dedicated starch store in its own right (in seeds and tubers), or as a temporary reserve of carbon contributing to sink strength, in organs such as flowers, fruits, and developing non-starchy seeds. The presence of starch in tissues and organs thus has a profound impact on the physiology of the growing plant as its synthesis and degradation governs the availability of free sugars, which in turn control various growth and developmental processes.

Resistant Starch Bagels Reduce Fasting and Postprandial Insulin in Adults at Risk of Type 2 Diabetes.

Background: Type 2 diabetes (T2D) incidence continues to rise. Although increasing dietary fiber intake is an established strategy for improved glycemic control, most adults consume insufficient amounts. Fiber-enhanced functional foods can increase fiber intake, and there is particular interest in resistant starch (RS) as a high-fiber ingredient.

Expression of Escherichia coli glycogen branching enzyme in an Arabidopsis mutant devoid of endogenous starch branching enzymes induces the synthesis of starch-like polyglucans.

Starch synthesis requires several enzymatic activities including branching enzymes (BEs) responsible for the formation of α(1 → 6) linkages. Distribution and number of these linkages are further controlled by debranching enzymes that cleave some of them, rendering the polyglucan water-insoluble and semi-crystalline. Although the activity of BEs and debranching enzymes is mandatory to sustain normal starch synthesis, the relative importance of each in the establishment of the plant storage polyglucan (i.

Second harmonic generation microscopy investigation of the crystalline ultrastructure of three barley starch lines affected by hydration.

Second harmonic generation (SHG) microscopy is employed to study changes in crystalline organization due to altered gene expression and hydration in barley starch granules. SHG intensity and susceptibility ratio values (R'SHG ) are obtained using reduced Stokes-Mueller polarimetric microscopy. The maximum R'SHG values occur at moderate moisture indicating the narrowest orientation distribution of nonlinear dipoles from the cylindrical axis of glucan helices.

Modification of starch metabolism in transgenic Arabidopsis thaliana increases plant biomass and triples oilseed production.

We have identified a novel means to achieve substantially increased vegetative biomass and oilseed production in the model plant Arabidopsis thaliana. Endogenous isoforms of starch branching enzyme (SBE) were substituted by either one of the endosperm-expressed maize (Zea mays L.) branching isozymes, ZmSBEI or ZmSBEIIb.

Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes.

Amylopectin is a highly branched, organized cluster of glucose polymers, and the major component of rice starch. Synthesis of amylopectin requires fine co-ordination between elongation of glucose polymers by soluble starch synthases (SSs), generation of branches by branching enzymes (BEs), and removal of misplaced branches by debranching enzymes (DBEs). Among the various isozymes having a role in amylopectin biosynthesis, limited numbers of SS and BE isozymes have been demonstrated to interact via protein-protein interactions in maize and wheat amyloplasts.

The different effects of starch synthase IIa mutations or variation on endosperm amylose content of barley, wheat and rice are determined by the distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma.

The distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma plays an important role in determining endosperm amylose content of cereal grains. Starch synthase IIa (SSIIa) catalyses the polymerisation of intermediate length glucan chains of amylopectin in the endosperm of cereals. Mutations of SSIIa genes in barley and wheat and inactive SSIIa variant in rice induce similar effects on the starch structure and the amylose content, but the severity of the phenotypes is different.

Protein-protein interactions among enzymes of starch biosynthesis in high-amylose barley genotypes reveal differential roles of heteromeric enzyme complexes in the synthesis of A and B granules.

The present study investigated the role of protein phosphorylation, and protein complex formation between key enzymes of amylopectin synthesis, in barley genotypes exhibiting "high amylose" phenotypes. Starch branching enzyme (SBE) down-regulated lines (ΔSBEIIa and ΔSBEIIb), starch synthase (SS)IIa (ssiia(-), sex6) and SSIII (ssiii(-), amo1) mutants were compared to a reference genotype, OAC Baxter. Down-regulation of either SBEIIa or IIb caused pleiotropic effects on SSI and starch phosphorylase (SP) and resulted in formation of novel protein complexes in which the missing SBEII isoform was substituted by SBEI and SP.

Second Harmonic Generation Mediated by Aligned Water in Starch Granules.

The origin of second harmonic generation (SHG) in starch granules was investigated using ab initio quantum mechanical modeling and experimentally examined using polarization-in, polarization-out (PIPO) second harmonic generation microscopy. Ab initio calculations revealed that the largest contribution to the SHG signal from A- and B-type allomorphs of starch originates from the anisotropic organization of hydroxide and hydrogen bonds mediated by aligned water found in the polymers. The hypothesis was experimentally tested by imaging maize starch granules under various hydration and heat treatment conditions that alter the hydrogen bond network.

A review of starch-branching enzymes and their role in amylopectin biosynthesis.

Starch-branching enzymes (SBEs) are one of the four major enzyme classes involved in starch biosynthesis in plants and algae, and their activities play a crucial role in determining the structure and physical properties of starch granules. SBEs generate α-1,6-branch linkages in α-glucans through cleavage of internal α-1,4 bonds and transfer of the released reducing ends to C-6 hydroxyls. Starch biosynthesis in plants and algae requires multiple isoforms of SBEs and is distinct from glycogen biosynthesis in both prokaryotes and eukaryotes which uses a single branching enzyme (BE) isoform.

Multimeric states of starch phosphorylase determine protein-protein interactions with starch biosynthetic enzymes in amyloplasts.

Protein-protein interactions between starch phosphorylase (SP) and other starch biosynthetic enzymes were investigated using isolated maize endosperm amyloplasts and a recombinant maize enzyme. Plastidial SP is a stromal enzyme existing as a multimeric protein in amyloplasts. Biochemical analysis of the recombinant maize SP indicated that the tetrameric form was catalytically active in both glucan-synthetic and phosphorolytic directions.

Identification of multiple phosphorylation sites on maize endosperm starch branching enzyme IIb, a key enzyme in amylopectin biosynthesis.

Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser(649), Ser(286), and Ser(297).

Resistant starch intake at breakfast affects postprandial responses in type 2 diabetics and enhances the glucose-dependent insulinotropic polypeptide--insulin relationship following a second meal.

Resistant starch (RS) consumption can modulate postprandial metabolic responses, but its effects on carbohydrate (CHO) handling in type 2 diabetics (T2D) are unclear. It was hypothesized that a bagel high in RS would improve glucose and insulin homeostasis following the 1st meal, regardless of the amount of available CHO, and that in association with incretins, the effects would carry over to a 2nd meal. Using a randomized crossover design, 12 T2D ingested four different bagel treatments (their 1st meal) determined by available CHO and the weight or amount of bagel consumed: treatment A, without RS (50 g of available CHO); treatment B, with RS (same total CHO as in A); treatment C, with RS (same available CHO as in A); and treatment D, with the same RS as in B and available CHO as in A and C.

Glucan affinity of starch synthase IIa determines binding of starch synthase I and starch-branching enzyme IIb to starch granules.

The sugary-2 mutation in maize (Zea mays L.) is a result of the loss of catalytic activity of the endosperm-specific SS (starch synthase) IIa isoform causing major alterations to amylopectin architecture. The present study reports a biochemical and molecular analysis of an allelic variant of the sugary-2 mutation expressing a catalytically inactive form of SSIIa and sheds new light on its central role in protein-protein interactions and determination of the starch granule proteome.

Allelic variants of the amylose extender mutation of maize demonstrate phenotypic variation in starch structure resulting from modified protein-protein interactions.

Amylose extender (ae(-)) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae(-) maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein-protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.

The amylose extender mutant of maize conditions novel protein-protein interactions between starch biosynthetic enzymes in amyloplasts.

The amylose extender (ae(-)) mutant of maize lacks starch branching enzyme IIb (SBEIIb) activity, resulting in amylopectin with reduced branch point frequency, and longer glucan chains. Recent studies indicate isozymes of soluble starch synthases form high molecular weight complexes with SBEII isoforms. This study investigated the effect of the loss of SBEIIb activity on interactions between starch biosynthetic enzymes in maize endosperm amyloplasts.

Oxidation of methionine residues: the missing link between stress and signalling responses in plants.

In response to biotic and abiotic stresses, plants induce a complex array of pathways and protein phosphorylation cascades which generally lead to a response aimed at mitigating the particular insult. In many cases, H2O2 has been implicated as the signalling molecule, but, although progress has been made in assembling the downstream components of these signalling pathways, far less is known about the mechanism by which the signal is perceived. In this issue of the Biochemical Journal, Hardin et al.

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm.

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an alpha-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm.

Starch biosynthetic enzymes from developing maize endosperm associate in multisubunit complexes.

Mutations affecting specific starch biosynthetic enzymes commonly have pleiotropic effects on other enzymes in the same metabolic pathway. Such genetic evidence indicates functional relationships between components of the starch biosynthetic system, including starch synthases (SSs), starch branching enzymes (BEs), and starch debranching enzymes; however, the molecular explanation for these functional interactions is not known. One possibility is that specific SSs, BEs, and/or starch debranching enzymes associate physically with each other in multisubunit complexes.

Analysis of protein complexes in wheat amyloplasts reveals functional interactions among starch biosynthetic enzymes.

Protein-protein interactions among enzymes of amylopectin biosynthesis were investigated in developing wheat (Triticum aestivum) endosperm. Physical interactions between starch branching enzymes (SBEs) and starch synthases (SSs) were identified from endosperm amyloplasts during the active phase of starch deposition in the developing grain using immunoprecipitation and cross-linking strategies. Coimmunoprecipitation experiments using peptide-specific antibodies indicate that at least two distinct complexes exist containing SSI, SSIIa, and either of SBEIIa or SBEIIb.

Characterization of ADP-glucose transport across the cereal endosperm amyloplast envelope.

Most of the carbon used for starch biosynthesis in cereal endosperms is derived from ADP-glucose (ADP-Glc) synthesized by extra-plastidial AGPase activity, and imported directly across the amyloplast envelope. The properties of the wheat endosperm amyloplast ADP-Glc transporter were analysed with respect to substrate kinetics and specificities using reconstituted amyloplast envelope proteins in a proteoliposome-based assay system, as well as with isolated intact organelles. Experiments with liposomes showed that ADP-Glc transport was dependent on counter-exchange with other adenylates.