Detecting variation in starch granule size and morphology by high-throughput microscopy and flow cytometry.

Starch forms semi-crystalline, water-insoluble granules, the size and morphology of which vary according to biological origin. These traits, together with polymer composition and structure, determine the physicochemical properties of starch. However, screening methods to identify differences in starch granule size and shape are lacking. Here, we present two approaches for high-throughput starch granule extraction and size determination using flow cytometry and automated, high-throughput light microscopy. We evaluated the practicality of both methods using starch from different species and tissues and demonstrated their effectiveness by screening for induced variation in starch extracted from over 10,000 barley lines, yielding four with heritable changes in the ratio of large A-granules to small B-granules. Analysis of Arabidopsis lines altered in starch biosynthesis further demonstrates the applicability of these approaches. Identifying variation in starch granule size and shape will enable identification of trait-controlling genes for developing crops with desired properties, and could help optimise starch processing.

FIND-IT: Accelerated trait development for a green evolution.

Improved agricultural and industrial production organisms are required to meet the future global food demands and minimize the effects of climate change. A new resource for crop and microbe improvement, designated FIND-IT (Fast Identification of Nucleotide variants by droplet DigITal PCR), provides ultrafast identification and isolation of predetermined, targeted genetic variants in a screening cycle of less than 10 days. Using large-scale sample pooling in combination with droplet digital PCR (ddPCR) greatly increases the size of low-mutation density and screenable variant libraries and the probability of identifying the variant of interest. The method is validated by screening variant libraries totaling 500,000 barley (Hordeum vulgare) individuals and isolating more than 125 targeted barley gene knockout lines and miRNA or promoter variants enabling functional gene analysis. FIND-IT variants are directly applicable to elite breeding pipelines and minimize time-consuming technical steps to accelerate the evolution of germplasm.

Identification and spatio-temporal expression analysis of barley genes that encode putative modular xylanolytic enzymes.

Arabinoxylans are cell wall polysaccharides whose re-modelling and degradation during plant development are mediated by several classes of xylanolytic enzymes. Here, we present the identification and new annotation of twelve putative (1,4)-ß-xylanase and six ß-xylosidase genes, and their spatio-temporal expression patterns during vegetative and reproductive growth of barley (Hordeum vulgare cv. Navigator). The encoded xylanase proteins are all predicted to contain a conserved carbohydrate-binding module (CBM) and a catalytic glycoside hydrolase (GH) 10 domain. Additional domains in some xylanases define three discrete phylogenetic clades: one clade contains proteins with an additional N-terminal signal sequence, while another clade contains proteins with multiple CBMs. Homology modelling revealed that all fifteen xylanases likely contain a third domain, a ß-sandwich folded from two non-contiguous sequence segments that bracket the catalytic GH domain, which may explain why the full length protein is required for correct folding of the active enzyme. Similarly, predicted xylosidase proteins share a highly conserved domain structure, each with an N-terminal signal peptide, a split GH 3 domain, and a C-terminal fibronectin-like domain. Several genes appear to be ubiquitously expressed during barley growth and development, while four newly annotated xylanase and xylosidase genes are expressed at extremely high levels, which may be of broader interest for industrial applications where cell wall degradation is necessary.

Genes That Mediate Starch Metabolism in Developing and Germinated Barley Grain.

Starch is synthesized in the endosperm of developing barley grain, where it functions as the primary source of stored carbohydrate. In germinated grain these starch reserves are hydrolyzed to small oligosaccharides and glucose, which are transported to the embryo to support the growth of the developing seedling. Some of the mobilized glucose is transiently stored as starch in the scutellum of germinated grain. These processes are crucial for early seedling vigor, which is a key determinant of crop productivity and global food security. Several starch synthases (SS), starch-branching enzymes (SBEs), and starch debranching enzymes (isoamylases, ISA), together with a limit dextrinase (LD), have been implicated in starch synthesis from nucleotide-sugar precursors. Starch synthesis occurs both in the developing endosperm and in the scutellum of germinated grain. For the complete depolymerization of starch to glucose, α-amylase (Amy), ß-amylase (Bmy), isoamylase (ISA), limit dextrinase (LD), and α-glucosidase (AGL) are required. Most of these enzymes are encoded by gene families of up to 10 or more members. Here RNA-seq transcription data from isolated tissues of intact developing and germinated barley grain have allowed us to identify the most important, specific gene family members for each of these processes in vivo and, at the same time, we have defined in detail the spatio-temporal coordination of gene expression in different tissues of the grain. A transcript dataset for 81,280 genes is publicly available as a resource for investigations into other cellular and biochemical processes that occur in the developing grain from 6 days after pollination.

Crystal Structures of the Catalytic Domain of Arabidopsis thaliana Starch Synthase IV, of Granule Bound Starch Synthase From CLg1 and of Granule Bound Starch Synthase I of Cyanophora paradoxa Illustrate Substrate Recognition in Starch Synthases.

Starch synthases (SSs) are responsible for depositing the majority of glucoses in starch. Structural knowledge on these enzymes that is available from the crystal structures of rice granule bound starch synthase (GBSS) and barley SSI provides incomplete information on substrate binding and active site architecture. Here we report the crystal structures of the catalytic domains of SSIV from Arabidopsis thaliana, of GBSS from the cyanobacterium CLg1 and GBSSI from the glaucophyte Cyanophora paradoxa, with all three bound to ADP and the inhibitor acarbose. The SSIV structure illustrates in detail the modes of binding for both donor and acceptor in a plant SS. CLg1GBSS contains, in the same crystal structure, examples of molecules with and without bound acceptor, which illustrates the conformational changes induced upon acceptor binding that presumably precede catalytic activity. With structures available from several isoforms of plant and non-plant SSs, as well as the closely related bacterial glycogen synthases, we analyze, at the structural level, the common elements that define a SS, the elements that are necessary for substrate binding and singularities of the GBSS family that could underlie its processivity. While the phylogeny of the SSIII/IV/V has been recently discussed, we now further report the detailed evolutionary history of the GBSS/SSI/SSII type of SSs enlightening the origin of the GBSS enzymes used in our structural analysis.

Combining mutations at genes encoding key enzymes involved in starch synthesis affects the amylose content, carbohydrate allocation and hardness in the wheat grain.

Modifications to the composition of starch, the major component of wheat flour, can have a profound effect on the nutritional and technological characteristics of the flour's end products. The starch synthesized in the grain of conventional wheats (Triticum aestivum) is a 3:1 mixture of the two polysaccharides amylopectin and amylose. Altering the activity of certain key starch synthesis enzymes (GBSSI, SSIIa and SBEIIa) has succeeded in generating starches containing a different polysaccharide ratio. Here, mutagenesis, followed by a conventional marker-assisted breeding exercise, has been used to generate three mutant lines that produce starch with an amylose contents of 0%, 46% and 79%. The direct and pleiotropic effects of the multiple mutation lines were identified at both the biochemical and molecular levels. Both the structure and composition of the starch were materially altered, changes which affected the functionality of the starch. An analysis of sugar and nonstarch polysaccharide content in the endosperm suggested an impact of the mutations on the carbon allocation process, suggesting the existence of cross-talk between the starch and carbohydrate synthesis pathways.

Functional and structural characterization of plastidic starch phosphorylase during barley endosperm development.

The production of starch is essential for human nutrition and represents a major metabolic flux in the biosphere. The biosynthesis of starch in storage organs like barley endosperm operates via two main pathways using different substrates: starch synthases use ADP-glucose to produce amylose and amylopectin, the two major components of starch, whereas starch phosphorylase (Pho1) uses glucose-1-phosphate (G1P), a precursor for ADP-glucose production, to produce α-1,4 glucans. The significance of the Pho1 pathway in starch biosynthesis has remained unclear. To elucidate the importance of barley Pho1 (HvPho1) for starch biosynthesis in barley endosperm, we analyzed HvPho1 protein production and enzyme activity levels throughout barley endosperm development and characterized structure-function relationships of HvPho1. The molecular mechanisms underlying the initiation of starch granule biosynthesis, that is, the enzymes and substrates involved in the initial transition from simple sugars to polysaccharides, remain unclear. We found that HvPho1 is present as an active protein at the onset of barley endosperm development. Notably, purified recombinant protein can catalyze the de novo production of α-1,4-glucans using HvPho1 from G1P as the sole substrate. The structural properties of HvPho1 provide insights into the low affinity of HvPho1 for large polysaccharides like starch or amylopectin. Our results suggest that HvPho1 may play a role during the initiation of starch biosynthesis in barley.

The down-regulation of the genes encoding Isoamylase 1 alters the starch composition of the durum wheat grain.

In rice, maize and barley, the lack of Isoamylase 1 activity materially affects the composition of endosperm starch. Here, the effect of this deficiency in durum wheat has been characterized, using transgenic lines in which Isa1 was knocked down via RNAi. Transcriptional profiling confirmed the partial down-regulation of Isa1 and revealed a pleiotropic effect on the level of transcription of genes encoding other isoamylases, pullulanase and sucrose synthase. The polysaccharide content of the transgenic endosperms was different from that of the wild type in a number of ways, including a reduction in the content of starch and a moderate enhancement of both phytoglycogen and ß-glucan. Some alterations were also induced in the distribution of amylopectin chain length and amylopectin fine structure. The amylopectin present in the transgenic endosperms was more readily hydrolyzable after a treatment with hydrochloric acid, which disrupted its semi-crystalline structure. The conclusion was that in durum wheat, Isoamylase 1 is important for both the synthesis of amylopectin and for determining its internal structure.

The Role of Cysteine Residues in Redox Regulation and Protein Stability of Arabidopsis thaliana Starch Synthase 1.

Starch biosynthesis in Arabidopsis thaliana is strictly regulated. In leaf extracts, starch synthase 1 (AtSS1) responds to the redox potential within a physiologically relevant range. This study presents data testing two main hypotheses: 1) that specific thiol-disulfide exchange in AtSS1 influences its catalytic function 2) that each conserved Cys residue has an impact on AtSS1 catalysis. Recombinant AtSS1 versions carrying combinations of cysteine-to-serine substitutions were generated and characterized in vitro. The results demonstrate that AtSS1 is activated and deactivated by the physiological redox transmitters thioredoxin f1 (Trxf1), thioredoxin m4 (Trxm4) and the bifunctional NADPH-dependent thioredoxin reductase C (NTRC). AtSS1 displayed an activity change within the physiologically relevant redox range, with a midpoint potential equal to -306 mV, suggesting that AtSS1 is in the reduced and active form during the day with active photosynthesis. Cys164 and Cys545 were the key cysteine residues involved in regulatory disulfide formation upon oxidation. A C164S_C545S double mutant had considerably decreased redox sensitivity as compared to wild type AtSS1 (30% vs 77%). Michaelis-Menten kinetics and molecular modeling suggest that both cysteines play important roles in enzyme catalysis, namely, Cys545 is involved in ADP-glucose binding and Cys164 is involved in acceptor binding. All the other single mutants had essentially complete redox sensitivity (98-99%). In addition of being part of a redox directed activity "light switch", reactivation tests and low heterologous expression levels indicate that specific cysteine residues might play additional roles. Specifically, Cys265 in combination with Cys164 can be involved in proper protein folding or/and stabilization of translated protein prior to its transport into the plastid. Cys442 can play an important role in enzyme stability upon oxidation. The physiological and phylogenetic relevance of these findings is discussed.

In vitro Biochemical Characterization of All Barley Endosperm Starch Synthases.

Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

Design of Glycosyltransferase Inhibitors: Serine Analogues as Pyrophosphate Surrogates?

Mimicking the diphosphate moiety of nucleotide diphosphate sugars with serine analogues provided modest glycosyltransferase inhibitors. The synthetic strategy employed a combination of glycosylation, amide bond formation and azide-alkyne "click" chemistry. Inhibition constants (Ki ) in the high micromolar range were obtained with a selection of five galactosyltransferases. Cocrystals of three inhibitors bound at the active site of a blood group A/B synthesizing glycosyltransferase were analysed. The structures and inhibitory patterns of the analogues demonstrate the flexibility of the enzymes which complicates the rational design of glycosyltransferase inhibitors.

Flexibility and mutagenic resiliency of glycosyltransferases.

The human blood group A and B antigens are synthesized by two highly homologous enzymes, glycosyltransferase A (GTA) and glycosyltransferase B (GTB), respectively. These enzymes catalyze the transfer of either GalNAc or Gal from their corresponding UDP-donors to αFuc1-2ßGal-R terminating acceptors. GTA and GTB differ at only four of 354 amino acids (R176G, G235S, L266M, G268A), which alter the donor specificity from UDP-GalNAc to UDP-Gal. Blood type O individuals synthesize truncated or non-functional enzymes. The cloning, crystallization and X-ray structure elucidations for GTA and GTB have revealed key residues responsible for donor discrimination and acceptor binding. Structural studies suggest that numerous conformational changes occur during the catalytic cycle. Over 300 ABO alleles are tabulated in the blood group antigen mutation database (BGMUT) that provides a framework for structure-function studies. Natural mutations are found in all regions of GTA and GTB from the active site, flexible loops, stem region and surfaces remote from the active site. Our characterizations of natural mutants near a flexible loop (V175M), on a remote surface site (P156L), in the metal binding motif (M212V) and near the acceptor binding site (L232P) demonstrate the resiliency of GTA and GTB to mutagenesis.

Design of glycosyltransferase inhibitors: pyridine as a pyrophosphate surrogate.

A series of ten glycosyltransferase inhibitors has been designed and synthesized by using pyridine as a pyrophosphate surrogate. The series was prepared by conjugation of carbohydrate, pyridine, and nucleoside building blocks by using a combination of glycosylation, the Staudinger-Vilarrasa amide-bond formation, and azide-alkyne click chemistry. The compounds were evaluated as inhibitors of five metal-dependent galactosyltransferases. Crystallographic analyses of three inhibitors complexed in the active site of one of the enzymes confirmed that the pyridine moiety chelates the Mn(2+) ion causing a slight displacement (2 Å) from its original position. The carbohydrate head group occupies a different position than in the natural uridine diphosphate (UDP)-Gal substrate with little interaction with the enzyme.

Structure of starch synthase I from barley: insight into regulatory mechanisms of starch synthase activity.

Starch, a polymer of glucose, is the major source of calories in the human diet. It has numerous industrial uses, including as a raw material for the production of first-generation bioethanol. Several classes of enzymes take part in starch biosynthesis, of which starch synthases (SSs) carry out chain elongation of both amylose and amylopectin. Plants have five classes of SS, each with different roles. The products of the reaction of SS are well known, but details of the reaction mechanism remain obscure and even less is known of how different SSs select different substrates for elongation, how they compete with each other and how their activities are regulated. Here, the first crystal structure of a soluble starch synthase is presented: that of starch synthase I (SSI) from barley refined to 2.7 Å resolution. The structure captures an open conformation of the enzyme with a surface-bound maltooligosaccharide and a disulfide bridge that precludes formation of the active site. The maltooligosaccharide-binding site is involved in substrate recognition, while the disulfide bridge is reflective of redox regulation of SSI. Activity measurements on several SSI mutants supporting these roles are also presented.

Continuous ß-turn fold of an alternating alanyl/homoalanyl peptide nucleic acid.

The crystal structure of the PNA (peptide nucleic acid) oligomer H-Lys-HalG-AlaG-HalC-AlaG-HalC-AlaC-Lys-NH(2) (PNA1, amino acids with D-configuration are underlined, Ala = alanyl, Hal = homoalanyl) has been determined by ab initio direct methods and refined against 1.0 Å data. The asymmetric unit consists of a tetrameric cage with almost ideal Watson-Crick C-G base pairing of all the guanine and cytosine side-chain substituents. Each PNA strand has a 90° ß-turn every second residue, stabilized by three hydrogen bonds between the backbone amides. The first, second, fifth and sixth bases stack on one side of the monomer and pair with the corresponding complementary bases of a second monomer to form a dimer. The two remaining bases on each side of the resulting dimer form Watson-Crick pairs with the complementary bases of a second dimer, leading to a unique cage structure. The extra methylene groups in the homoalanyl residues enable stacking of the bases with an optimal distance between base-planes but also with an appreciable lateral displacement (slide).

Interaction of an echinomycin-DNA complex with manganese ions.

The crystal structure of an echinomycin-d(ACGTACGT) duplex interacting with manganese(II) was solved by Mn-SAD using in-house data and refined to 1.1 A resolution against synchrotron data. This complex crystallizes in a different space group compared with related complexes and shows a different mode of base pairing next to the bis-intercalation site, suggesting that the energy difference between Hoogsteen and Watson-Crick pairing is rather small. The binding of manganese to N7 of guanine is only possible because of DNA unwinding induced by the echinomycin, which might help to explain the mode of action of the drug.

Crystal structure of a self-assembling lipopeptide detergent at 1.20 A.

Lipopeptide detergents (LPDs) are a new class of amphiphile designed specifically for the structural study of integral membrane proteins. The LPD monomer consists of a 25-residue peptide with fatty acyl chains linked to side chains located at positions 2 and 24 of the peptide. LPDs are designed to form alpha-helices that self-assemble into cylindrical micelles, providing a more natural interior acyl chain packing environment relative to traditional detergents. We have determined the crystal structure of LPD-12, an LPD coupled to two dodecanoic acids, to a resolution of 1.20 A. The LPD-12 monomers adopt the target conformation and associate into cylindrical octamers as expected. Pairs of helices are strongly associated as Alacoil-type antiparallel dimers, and four of these dimers interact through much looser contacts into assemblies with approximate D(2) symmetry. The aligned helices form a cylindrical shell with a hydrophilic exterior that protects an interior hydrophobic cavity containing the 16 LPD acyl chains. Over 90% of the methylene/methyl groups from the acylated side chains are visible in the micelle interiors, and approximately 90% of these adopt trans dihedral angle conformations. Dodecylmaltoside (DDM) was required for the crystallization of LPD-12, and we find 10-24 ordered DDM molecules associated with each LPD assembly, resulting in an overall micelle molecular weight of approximately 30 kDa. The structures confirm the major design objectives of the LPD framework, and reveal unexpected features that will be helpful in the engineering additional versions of lipopeptide amphiphiles.

Serendipitous SAD phasing of an echinomycin-(ACGTACGT)2 bisintercalation complex.

A new crystal form was obtained for the complex between (ACGTACGT)2 and echinomycin and X-ray data were collected to 1.6 A. The structure was phased by the SAD method based on a single unexpected anomalous scatterer that could be identified as a mixture of nickel and zinc by measurements of the anomalous scattering at different wavelengths. This cation is coordinated by two guanines from two different duplexes and four water molecules. The structure resembles previously reported crystal structures of DNA-echinomycin complexes, except that three of the eight base pairs flanking the echinomycin bisintercalator sites have the Watson-Crick rather than the Hoogsteen configuration. Hoogsteen binding was found for the corresponding base pairs of the crystallographically independent duplex, indicating that the two configurations are very close in energy.

Structures of complexes between echinomycin and duplex DNA.

The structure of the bis-intercalation complex of the depsipeptide antibiotic echinomycin with (CGTACG)2 has been redetermined at a higher resolution (1.4 A) and new high-resolution structures (1.1-1.5 A) are reported for the complexes of echinomycin with (GCGTACGC)2 (at both low and high ionic strengths) and (ACGTACGT)2. The structures show the expected Hoogsteen pairing for the base pairs flanking the intercalating chromophores on the outside and Watson-Crick pairing for both base pairs enclosed by the echinomycin. In the octamer complexes but not the hexamer complex, the echinomycin molecule, which would possess a molecular twofold axis were it not for the thioacetal bridge, shows twofold disorder. In all the structures the stacking of the base pairs and chromophores is extended by intermolecular stacking. The structures provide more precise details of the hydrogen bonding and other interactions between the bis-intercalating antibiotics and the duplex DNA than were previously available.