Integrated functions among multiple starch synthases determine both amylopectin chain length and branch linkage location in Arabidopsis leaf starch

This study assessed the impact on starch metabolism in Arabidopsis leaves of simultaneously eliminating multiple soluble starch synthases (SS) from among SS1, SS2, and SS3. Double mutant ss1- ss2- or ss1- ss3- lines were generated using confirmed null mutations. These were compared to the wild type, each single mutant, and ss1- ss2- ss3- triple mutant lines grown in standardized environments. Double mutant plants developed similarly to the wild type, although they accumulated less leaf starch in both short-day and long-day diurnal cycles. Despite the reduced levels in the double mutants, lines containing only SS2 and SS4, or SS3 and SS4, are able to produce substantial amounts of starch granules. In both double mutants the residual starch was structurally modified including higher ratios of amylose:amylopectin, altered glucan chain length distribution within amylopectin, abnormal granule morphology, and altered placement of α(1→6) branch linkages relative to the reducing end of each linear chain. The data demonstrate that SS activity affects not only chain elongation but also the net result of branch placement accomplished by the balanced activities of starch branching enzymes and starch debranching enzymes. SS3 was shown partially to overlap in function with SS1 for the generation of short glucan chains within amylopectin. Compensatory functions that, in some instances, allow continued residual starch production in the absence of specific SS classes were identified, probaby accomplished by the granule bound starch synthase GBSS1.ANR Génoplante GPLA0611GEuropean Union-FEDER, Région Nord Pas de Calais ARCir PlantTEQ5National Science Foundation DBI-0209789Comisión Interministerial de Ciencia y Tecnología BIO2009-07040Junta de Andalucía P09-CVI-470

Production of amylopectin and high-amylose starch in separate potato genotypes

Starch is one of the most important processed products from agriculture. Two main outlets can be identified; starch is either enzymatically processed for the production of sweeteners and as raw material for fermentation or channelled to various applications as dry starch. Native or chemically modified starches are utilized in food as well as non-food applications, where the specific physicochemical properties are main determinants for their respective use. Starch consists of two different molecules, amylose and amylopectin. To be able to take the full benefit of the unique properties of either component it is of interest to divide the production of amylose and amylopectin into separate plant genotypes. In the presented work, potatoes producing either amylopectin or high-amylose starch were achieved using genetic modification. For potato transformation a highly efficient protocol was developed for a herbicide selection gene instead of the commonly used nptII antibiotic selection gene. In order to achieve respective starch qualities, the expression of genes important for amylopectin or amylose synthesis was silenced. Antisense technology as well as the expression of dsRNA was investigated where the expression of dsRNA was determined to be at least ten-fold more efficient for gene silencing. An added benefit of dsRNA expression was that a higher fraction of silenced transgenic lines compared to the use antisense were associated with single copy T-DNA integrations. One amylopectin potato line was furthermore characterized regarding genetic and chemical composition. The T-DNA was found integrated as an inverted repeat with the inverted repeat region extending into potato chromosomal DNA. This transgenic locus was found to be more consistent with integration into a double-stranded chromosomal break than insertion by a mechanism nicking one strand of the locus. The high-amylose trait generally resulted in a higher tuber fresh weight yield, much elevated sugar levels and a decreased starch content. Amylose levels were obtained where very limited amounts of material recognizable as amylopectin could be found. The production of amylopectin and amylose was divided into separate genotypes but additional factors are needed to be able to produce amylose at levels comparable to starch contents of cultivated potatoes

Different genetic strategies to generate high amylose starch mutants by engineering the starch biosynthetic pathways

This review systematically documents the major different strategies of generating high-amylose (HAS) starch mutants aiming at providing high resistant starch, by engineering the starch biosynthesis metabolic pathways. We identify three main strategies based on a new representation of the starch structure: 'the building block backbone model': i) suppression of starch synthases for reduction of amylopectin (AP) side-chains; ii) suppression of starch branching enzymes (SBEs) for production of AM-like materials; and iii) suppression of debranching enzymes to restrain the transformation from over-branched pre-AP to more ordered AP. From a biosynthetic perspective, AM generated through the second strategy can be classified into two types: i) normal AM synthesized mainly by regular expression of granule-bound starch synthases, and ii) modified linear AP chains (AM-like material) synthesized by starch synthases due to the suppression of starch branching enzymes. The application of new breeding technologies, especially CRISPR, in the breeding of HAS crops is also reviewed

The structure-function relationships of maize starch synthase

This research was to study the structure-function relationships and catalytic mechanisms of maize starch synthase IIa (SSIIa). Chemical modification of maize SSIIa showed that pyridoxal-5-phosphate, which specifically modifies lysine residues, inactivated maize SSIIa activity in a time and concentration dependent manner. Substrate ADP-glucose completely protected SSIIa from inactivation by pyridoxal-5-phosphate, indicating that lysine residue(s) is important for ADP-glucose binding of maize SSIIa. To identify lysine residues possibly involved in substrate ADP-glucose binding, site-directed mutagenesis was used to generate mutants at conserved lysine sites. Lysine-193 located at the conserved KTGGL domain has been widely suggested as a putative ADP-glucose binding site in plants based on the study of E. coli glycogen synthase. In contrast with E. coli glycogen synthase (GS), the mutations at the conserved lysine-193 did not change the ADP-glucose affinity of maize SSIIa. It suggests that the epsilon-amino group of lysine-193 is not involved in the binding of ADP-glucose in maize SSIIa. However, the mutations at lysine-193 influenced enzyme activity of maize SSIIa, suggesting that lysine-193 is involved in catalysis of maize SSIIa. The functional difference in the conserved K-T-G-G motif between E. coli GS and maize SSIIa is not related to the N-terminal extension of SSIIa, a structural difference between the two enzymes. Kinetic characterization of mutants at lysine-497 has shown that K497Q, K497N, and K497E resulted in a significant increase in Km for ADP-glucose over wild type enzyme. The kinetic changes are not caused by a global conformational change, as shown by Circular Dichroism spectra. This suggests that the conserved lysine-497 may be the ADP-glucose binding site of maize SSIIa. A three-dimensional structure model of maize SSIIa has been proposed based on protein threading and site-directed mutagenesis. In this model, the likely scenario of catalysis and substrate binding in maize SSIIa is that lysine-193 is involved in catalysis, and lysine-497 and aspartic acid 199 participate in substrate ADP-glucose binding of SSIIa. Furthermore, my research also showed that citrate not only increased primer affinity to the enzyme, but also affected catalytic chain elongation specificity of maize SSIIa. These findings may lead to generating improved starch synthases through protein engineering. Subsequent transformation of these enzymes in commercial crops may improve starch quantity and quality

<i>In vitro</i> 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

Characterization of function of the GlgA2 glycogen/starch synthase in Cyanobacterium sp. Clg1 highlights convergent evolution of glycogen metabolism into starch granule aggregation

At variance with the starch-accumulating plants and most of the glycogen-accumulating cyanobacteria, Cyanobacterium sp. CLg1 synthesizes both glycogen and starch. We now report the selection of a starchless mutant of this cyanobacterium that retains wild-type amounts of glycogen. Unlike other mutants of this type found in plants and cyanobacteria, this mutant proved to be selectively defective for one of the two types of glycogen/starch synthase: GlgA2. This enzyme is phylogenetically related to the previously reported SSIII/SSIV starch synthase that is thought to be involved in starch granule seeding in plants. This suggests that, in addition to the selective polysaccharide debranching demonstrated to be responsible for starch rather than glycogen synthesis, the nature and properties of the elongation enzyme define a novel determinant of starch versus glycogen accumulation. We show that the phylogenies of GlgA2 and of 16S ribosomal RNA display significant congruence. This suggests that this enzyme evolved together with cyanobacteria when they diversified over 2 billion years ago. However, cyanobacteria can be ruled out as direct progenitors of the SSIII/SSIV ancestral gene found in Archaeplastida. Hence, both cyanobacteria and plants recruited similar enzymes independently to perform analogous tasks, further emphasizing the importance of convergent evolution in the appearance of starch from a preexisting glycogen metabolism network.Peer Reviewe

Development of genome-specific primers for homoeologous genes in allopolyploid species: the waxy and starch synthase II genes in allohexaploid wheat (Triticum aestivum L.) as examples

<p>Abstract</p> <p>Background</p> <p>In allopolypoid crops, homoeologous genes in different genomes exhibit a very high sequence similarity, especially in the coding regions of genes. This makes it difficult to design genome-specific primers to amplify individual genes from different genomes. Development of genome-specific primers for agronomically important genes in allopolypoid crops is very important and useful not only for the study of sequence diversity and association mapping of genes in natural populations, but also for the development of gene-based functional markers for marker-assisted breeding. Here we report on a useful approach for the development of genome-specific primers in allohexaploid wheat.</p> <p>Findings</p> <p>In the present study, three genome-specific primer sets for the <it>waxy </it>(<it>Wx</it>) genes and four genome-specific primer sets for the <it>starch synthase II </it>(<it>SSII</it>) genes were developed mainly from single nucleotide polymorphisms (SNPs) and/or insertions or deletions (Indels) in introns and intron-exon junctions. The size of a single PCR product ranged from 750 bp to 1657 bp. The total length of amplified PCR products by these genome-specific primer sets accounted for 72.6%-87.0% of the <it>Wx </it>genes and 59.5%-61.6% of the <it>SSII </it>genes. Five genome-specific primer sets for the <it>Wx </it>genes (one for Wx-7A, three for Wx-4A and one for Wx-7D) could distinguish the wild type wheat and partial waxy wheat lines. These genome-specific primer sets for the <it>Wx </it>and <it>SSII </it>genes produced amplifications in hexaploid wheat, cultivated durum wheat, and <it>Aegilops tauschii </it>accessions, but failed to generate amplification in the majority of wild diploid and tetraploid accessions.</p> <p>Conclusions</p> <p>For the first time, we report on the development of genome-specific primers from three homoeologous <it>Wx </it>and <it>SSII </it>genes covering the majority of the genes in allohexaploid wheat. These genome-specific primers are being used for the study of sequence diversity and association mapping of the three homoeologous <it>Wx </it>and <it>SSII </it>genes in natural populations of both hexaploid wheat and cultivated tetraploid wheat. The strategies used in this paper can be used to develop genome-specific primers for homoeologous genes in any allopolypoid species. They may be also suitable for (i) the development of gene-specific primers for duplicated paralogous genes in any diploid species, and (ii) the development of allele-specific primers at the same gene locus.</p

Genetic and molecular analysis of starch synthases functions in maize and Arabidopsis

Understanding the specific functions played by individual starch synthase isoforms in maize and Arabidopsis will provide important evidence for how highly organized starch structure is made. Starch synthases (SS) catalyze the transfer of the glucosyl moiety from ADP-Glc to the terminus of a growing alpha-(1, 4)-linked glucan linear chain. At least five classes of SSs are identified in higher species, referred to as GBSS, SSI, SSII, SSIII, and SSIVN. They have high similarity in the catalytic and starch-binding domains of the C-termini but differ at their N-termini. All of these enzymes are highly conserved in plant kingdom, which indicates that they might have specific functions during starch biosynthesis;To investigate the functions of SSII, changes in starch biosynthesis caused by mutation of the sugary 2 (su2) gene in maize, two allelic su2-mutations were characterized. Su2 was shown to code for SSIIa, and both mutant alleles cause loss of SSIIa activity. Starch was characterized with respect to structural changes to the amylopectin (Ap) component of starch granules, changes of the amylopectin:amylose (Am) compositional ratio, and pleiotropic effects on other starch metabolizing enzymes. Loss of SSII resulted in Ap with more short chains of degree of polymerization (DP) 5-11 and fewer intermediate chains of DP12-25. Increased Am:Ap ratio was also observed in the mutant starches. The changes in Ap structure and Am:Ap ratio are similar to the effects of SSII deficiency in other species. The results demonstrate that some function of SSII is necessary for the normal accumulation of chains of DP12-25 in Ap;A similar approach was used in Arabidopsis to study the function of both SSII and SSIII. Six SS genes were identified in the Arabidopsis genome. To examine the specific functions of different SS isoforms or the functional interactions among them, either single mutations or a combination of double mutations in SS genes were studied. (Abstract shortened by UMI.