Stochastic modelling of a three-dimensional glycogen granule synthesis and impact of the branching enzyme.

In humans, glycogen storage diseases result from metabolic inborn errors, and can lead to severe phenotypes and lethal conditions. Besides these rare diseases, glycogen is also associated to widely spread societal burdens such as diabetes. Glycogen is a branched glucose polymer synthesised and degraded by a complex set of enzymes. Over the past 50 years, the structure of glycogen has been intensively investigated. Yet, the interplay between the detailed three-dimensional glycogen structure and the related enzyme activity is only partially characterised and still to be fully understood. In this article, we develop a stochastic coarse-grained and spatially resolved model of branched polymer biosynthesis following a Gillespie algorithm. Our study largely focusses on the role of the branching enzyme, and first investigates the properties of the model with generic parameter values, before comparing it to in vivo experimental data in mice. It arises that the ratio of glycogen synthase over branching enzyme reaction rates drastically impacts the structure of the granule. We deeply investigate the mechanism of branching and parametrise it using distinct lengths. Not only do we consider various possible sets of values for these lengths, but also distinct rules to apply them. We show how combining various values for these lengths finely tunes glycogen macromolecular structure. Comparing the model with experimental data confirms that we can accurately reproduce glycogen chain length distributions in wild type mice. Additional granule properties obtained for this fit are also in good agreement with typically reported values in the experimental literature. Nonetheless, we find that the mechanism of branching must be more flexible than usually reported. Overall, our model provides a theoretical basis to quantify the effect that single enzymatic parameters, in particular of the branching enzyme, have on the chain length distribution. Our generic model and methods can be applied to any glycogen data set, and could in particular contribute to characterise the mechanisms responsible for glycogen storage disorders.

Characterization of Two Glycoside Hydrolases of Family GH13 and GH57, Present in a Polysaccharide Utilization Locus (PUL) of Pontibacter sp. SGAir0037.

Glycogen, an α-glucan polymer serving as an energy storage compound in microorganisms, is synthesized through distinct pathways (GlgC-GlgA or GlgE pathway). Both pathways involve multiple enzymes, with a shared glycogen branching enzyme (GBE). GBEs play a pivotal role in establishing α-1,6-linkages within the glycogen structure. GBEs are also used for starch modification. Understanding how these enzymes work is interesting for both glycogen synthesis in microorganisms, as well as novel applications for starch modification. This study focuses on a putative enzyme GH13_9 GBE (PoGBE13), present in a polysaccharide utilization locus (PUL) of Pontibacter sp. SGAir0037, and related to the GlgE glycogen synthesis pathway. While the PUL of Pontibacter sp. SGAir0037 contains glycogen-degrading enzymes, the branching enzyme (PoGBE13) was also found due to genetic closeness. Characterization revealed that PoGBE13 functions as a typical branching enzyme, exhibiting a relatively high branching over non-branching (hydrolysis and α-1,4-transferase activity) ratio on linear maltooctadecaose (3.0 ± 0.4). Besides the GH13_9 GBE, a GH57 (PoGH57) enzyme was selected for characterization from the same PUL due to its undefined function. The combined action of both GH13 and GH57 enzymes suggested 4-α-glucanotransferase activity for PoGH57. The characterization of these unique enzymes related to a GlgE glycogen synthesis pathway provides a more profound understanding of their interactions and synergistic roles in glycogen synthesis and are potential enzymes for use in starch modification processes. Due to the structural similarity between glycogen and starch, PoGBE13 can potentially be used for starch modification with different applications, for example, in functional food ingredients.

A model for the reproduction of amylopectin cluster by coordinated actions of starch branching enzyme isoforms.

Amylopectin is a highly branched glucan which accounts for approximately 65-85% of starch in most plant tissues. It is crucially important to understand the biosynthetic process of this glucan in regulating the structure and functional properties of starch granules. Currently, the most accepted ideas of structural feature and biosynthesis of amylopectin are that amylopectin is composed of a branched element called "cluster" and that the essential process of amylopectin biosynthesis is to reproduce a new cluster from the existing cluster. The present paper proposes a model explaining the whole process of amylopectin biosynthesis as to how the new cluster is reproduced by concerted actions of multiple isoforms of starch biosynthetic enzymes, particularly by combinations of distinct roles of starch branching enzyme (BE) isoforms. This model proposes for the first time the molecular mechanism as to how the formation of a new cluster is initiated, and the reason why BEI can play a major role in this step. This is because BEI has a rather broad chain-length preference compared to BEIIb, because a low preference of BEI for the substrate chain-length is advantageous for branching a couple of elongated chains that are not synchronously formed and thus these chains having varied lengths could be safely attacked by this isoform. On the contrary, it is unlikely that BEIIb is involved in this reaction because it can react to only short chains having degree of polymerization of 12-14. BEIIa is possibly able to complement the role of BEI to some extent, because BEIIa can attack basically short chains but its chain-length preference is lower compared with BEIIb. The model implies that the first branches mainly formed by BEI to construct the amorphous lamellae whereas the second branches predominantly formed by BEIIb are located mainly in the crystalline lamellae. This paper provides new insights into the roles of BEI, BEIIb, and BEIIa in amylopectin biosynthesis in cereal endosperm.

Abundant copathologies of polyglucosan bodies, frontotemporal lobar degeneration with TDP-43 inclusions and ageing-related tau astrogliopathy in a family with a GBE1 mutation.

AIMS: Adult polyglucosan body disease (APBD) is a progressive neurogenetic disorder caused by 1,4-alpha-glucan branching enzyme 1 (GBE1) mutation with an accumulation of polyglucosan bodies (PBs) in the central and peripheral nervous systems as a pathological hallmark. Here, we report two siblings in a family with a GBE1 mutation with prominent frontotemporal lobar degeneration with TAR DNA-binding protein 43 (FTLD-TDP) and ageing-related tau astrogliopathy (ARTAG) copathologies with PBs in the central nervous system. METHODS: Whole-genome sequencing (WGS) followed by Sanger sequencing (SS) was performed on three affected and two unaffected siblings in a pedigree diagnosed with familial frontotemporal dementia. Out of the affected siblings, autopsies were conducted on two cases, and brain samples were used for biochemical and histological analyses. Brain sections were stained with haematoxylin and eosin and immunostained with antibodies against ubiquitin, tau, amyloid ß, α-synuclein, TDP-43 and fused in sarcoma (FUS). RESULTS: A novel single nucleotide deletion in GBE1, c.1280delG, was identified, which is predicted to result in a reading frameshift, p.Gly427Glufs*9. This variant segregated with disease in the family, is absent from population databases and is predicted to cause loss of function, a known genetic mechanism for APBD. The affected siblings showed a greater than 50% decrease in GBE protein levels. Immunohistochemical analysis revealed widespread FTLD-TDP (type A) and ARTAG pathologies as well as PBs in the brains of two affected siblings for whom an autopsy was performed. CONCLUSIONS: This is the first report of a family with several individuals with a FTD clinical phenotype and underlying copathologies of APBD, FTLD-TDP and ARTAG with a segregating GBE1 loss-of-function mutation in affected siblings. The finding of copathologies of APBD and FTLD-TDP suggests these processes may share a disease mechanism resulting from this GBE1 mutation.

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. All mutations were heterozygous monoallelic or biallelic, and no chimeric mutations were detected from a total of 216 editing events. Previously unannotated gene duplication events associated with two BnaSBE genes were characterized through analysis of DNA sequencing chromatograms, reflecting the complexity of genetic information in B. napus. Five Cas9-free homozygous mutant lines carrying two to six mutations of BnaSBE were obtained, allowing us to compare the effect of editing different BnaSBE isoforms. We also found that in the sextuple sbe mutant, although indels were introduced at the genomic DNA level, an alternate transcript of one BnaSBE2.1 gene bypassed the indel-induced frame shift and was translated to a modified full-length protein. Subsequent analyses showed that the sextuple mutant possesses much lower SBE enzyme activity and starch branching frequency, higher starch-bound phosphate content, and altered pattern of amylopectin chain length distribution relative to wild-type (WT) plants. In the sextuple mutant, irregular starch granules and a slower rate of starch degradation during darkness were observed in rosette leaves. At the pod-filling stage, the sextuple mutant was distinguishable from WT plants by its thick main stem. This work demonstrates the applicability of the CRISPR-Cas9 system for the study of multi-gene families and for investigation of gene-dosage effects in the oil crop B. napus. It also highlights the need for rigorous analysis of CRISPR-Cas9-mutated plants, particularly with higher levels of ploidy, to ensure detection of gene duplications.

Inactivation of rice starch branching enzyme IIb triggers broad and unexpected changes in metabolism by transcriptional reprogramming.

Starch properties can be modified by mutating genes responsible for the synthesis of amylose and amylopectin in the endosperm. However, little is known about the effects of such targeted modifications on the overall starch biosynthesis pathway and broader metabolism. Here we investigated the effects of mutating the OsSBEIIb gene encoding starch branching enzyme IIb, which is required for amylopectin synthesis in the endosperm. As anticipated, homozygous mutant plants, in which OsSBEIIb was completely inactivated by abolishing the catalytic center and C-terminal regulatory domain, produced opaque seeds with depleted starch reserves. Amylose content in the mutant increased from 19.6 to 27.4% and resistant starch (RS) content increased from 0.2 to 17.2%. Many genes encoding isoforms of AGPase, soluble starch synthase, and other starch branching enzymes were up-regulated, either in their native tissues or in an ectopic manner, whereas genes encoding granule-bound starch synthase, debranching enzymes, pullulanase, and starch phosphorylases were largely down-regulated. There was a general increase in the accumulation of sugars, fatty acids, amino acids, and phytosterols in the mutant endosperm, suggesting that intermediates in the starch biosynthesis pathway increased flux through spillover pathways causing a profound impact on the accumulation of multiple primary and secondary metabolites. Our results provide insights into the broader implications of perturbing starch metabolism in rice endosperm and its impact on the whole plant, which will make it easier to predict the effect of metabolic engineering in cereals for nutritional improvement or the production of valuable metabolites.

Characterization of the Glucan-Branching Enzyme GlgB Gene from Swine Intestinal Bacteria.

Starch hydrolysis by gut microbiota involves a diverse range of different enzymatic activities. Glucan-branching enzyme GlgB was identified as the most abundant glycosidase in Firmicutes in the swine intestine. GlgB converts α-(1→4)-linked amylose to form α-(1→4,6) branching points. This study aimed to characterize GlgB cloned from a swine intestinal metagenome and to investigate its potential role in formation of α-(1→4,6)-branched α-glucans from starch. The branching activity of purified GlgB was determined with six different starches and pure amylose by quantification of amylose after treatment. GlgB reduced the amylose content of all 6 starches and amylose by more than 85% and displayed a higher preference towards amylose. The observed activity on raw starch indicated a potential role in the primary starch degradation in the large intestine as an enzyme that solubilizes amylose. The oligosaccharide profile showed an increased concentration of oligosaccharide introduced by GlgB that is not hydrolyzed by intestinal enzymes. This corresponded to a reduced in vitro starch digestibility when compared to untreated starch. The study improves our understanding of colonic starch fermentation and may allow starch conversion to produce food products with reduced digestibility and improved quality.

A family study implicates GBE1 in the etiology of autism spectrum disorder.

Autism spectrum disorders (ASD) are neurodevelopmental disorders with an estimated heritability of >60%. Family-based genetic studies of ASD have generally focused on multiple small kindreds, searching for de novo variants of major effect. We hypothesized that molecular genetic analysis of large multiplex families would enable the identification of variants of milder effects. We studied a large multigenerational family of European ancestry with multiple family members affected with ASD or the broader autism phenotype (BAP). We identified a rare heterozygous variant in the gene encoding 1,4-ɑ-glucan branching enzyme 1 (GBE1) that was present in seven of seven individuals with ASD, nine of ten individuals with the BAP, and none of four tested unaffected individuals. We genotyped a community-acquired cohort of 389 individuals with ASD and identified three additional probands. Cascade analysis demonstrated that the variant was present in 11 of 13 individuals with familial ASD/BAP and neither of the two tested unaffected individuals in these three families, also of European ancestry. The variant was not enriched in the combined UK10K ASD cohorts of European ancestry but heterozygous GBE1 deletion was overrepresented in large ASD cohorts, collectively suggesting an association between GBE1 and ASD.

The Candida glabrata glycogen branching enzyme structure reveals unique features of branching enzymes of the Saccharomycetaceae phylum.

Branching enzymes (BE) are responsible for the formation of branching points at the 1,6 position in glycogen and starch, by catalyzing the cleavage of α-1,4-linkages and the subsequent transfer by introducing α-1,6-linked glucose branched points. BEs are found in the large GH13 family, eukaryotic BEs being mainly classified in the GH13_8 subfamily, GH13_9 grouping almost exclusively prokaryotic enzymes. With the aim of contributing to the understanding of the mode of recognition and action of the enzymes belonging to GH13_8, and to the understanding of features distinguishing these enzymes from those belonging to subfamily 13_9, we solved the crystal structure of the glycogen branching enzyme (GBE) from the yeast Candida glabrata, CgGBE, in ligand-free forms and in complex with a maltotriose. The structures revealed the presence of a domain already observed in Homo sapiens and Oryza sativa BEs that we named α-helical N-terminal domain, in addition to the three conserved domains found in BE. We confirmed by phylogenetic analysis that this α-helical N-terminal domain is always present in the GH13_8 enzymes suggesting that it could actually present a signature for this subfamily. We identified two binding sites in the α-helical N-terminal domain and in the carbohydrate binding module 48 (CBM48), respectively, which show a unique structural organization only present in the Saccharomycotina phylum. Our structural and phylogenetic investigation provides new insight into the structural characterization of GH13_8 GBE revealing that unique structural features only present in the Saccharomycotina phylum thereby conferring original properties to this group of enzymes.

Mutation in BEIIb mitigates the negative effect of the mutation in ISA1 on grain filling and amyloplast formation in rice.

KEY MESSAGE: Mutation of the BEIIb gene in an isa1 mutant background mitigates the negative effect of the ISA1 mutation on grain filling, and facilitates recovery of amyloplast formation in rice endosperm. In this study, the effect of branching enzyme IIb and isoamylase 1 deficiency on starch properties was demonstrated using high resistant starch rice lines, Chikushi-kona 85 and EM129. Both lines harbored a mutation in the BEIIb and ISA1 genes and showed no BEIIb and ISA1 activity, implying that both lines are beIIb isa1 double mutants. The amylopectin long chain and apparent amylose content of both mutant lines were higher than those of the wild-type. While both mutants contained loosely packed, round starch grains, a trait specific to beIIb mutants, they also showed collapsed starch grains at the center of the endosperm, a property specific to isa1 mutants. Furthermore, beIIb isa1 double mutant F2 lines derived from a cross between Chikushi-kona 85 and Nishihomare (wild-type cultivar) showed significantly heavier seed weight than the beIIb and isa1 single mutant lines. These results suggest that co-occurrence of beIIb and isa1 mutant alleles in a single genetic background mitigates the negative effect of the isa1 allele on grain filling, and contributes to recovery of the amyloplast formation defect in the isa1 single mutant.

Suppressed expression of starch branching enzyme 1 and 2 increases resistant starch and amylose content and modifies amylopectin structure in cassava.

KEY MESSAGE: Suppression of starch branching enzymes 1 and 2 in cassava leads to increased resistant starch content through the production of high-amylose and modification of the amylopectin structure. Cassava (Manihot esculenta Crantz) is a starchy root crop used for human consumption as a staple food and industrial applications. Starch is synthesized by various isoforms of several enzymes. However, the function of starch branching enzymes (SBEs) in starch biosynthesis and mechanisms of starch regulation in cassava have not been understood well. In this study, we aimed to suppress the expression of SBEs in cassava to generate starches with a range of distinct properties, in addition to verifying the functional characteristics of the SBEs. One SBE1, two SBE2, and one SBE3 genes were classified by phylogenetic analysis and amino acid alignment. Quantitative real-time RT-PCR revealed tissue-specific expression of SBE genes in the tuberous roots and leaves of cassava. We introduced RNAi constructs containing fragments of SBE1, SBE2, or both genes into cassava by Agrobacterium-mediated transformation, and assessed enzymatic activity of SBE using tuberous roots and leaves from these transgenic plants. Simultaneous suppression of SBE1 and SBE2 rendered an extreme starch phenotype compared to suppression of SBE2 alone. Degree of polymerization of 6-13 chains in amylopectin was markedly reduced by suppression of both SBE1 and SBE2 in comparison to the SBE2 suppression; however, no change in chain-length profiles was observed in the SBE1 suppression alone. The role of SBE1 and SBE2 may have functional overlap in the storage tissue of cassava. Simultaneous suppression of SBE1 and SBE2 resulted in highly resistant starch with increased apparent amylose content compared to suppression of SBE2 alone. This study provides valuable information for understanding starch biosynthesis and suggests targets for altering starch quality.

Editing of the starch branching enzyme gene SBE2 generates high-amylose storage roots in cassava.

KEY MESSAGE: The production of high-amylose cassava through CRISPR/Cas9-mediated mutagenesis of the starch branching enzyme gene SBE2 was firstly achieved. High-amylose cassava (Manihot esculenta Crantz) is desirable for starch industrial applications and production of healthier processed food for human consumption. In this study, we report the production of high-amylose cassava through CRISPR/Cas9-mediated mutagenesis of the starch branching enzyme 2 (SBE2). Mutations in two targeted exons of SBE2 were identified in all regenerated plants; these mutations, which included nucleotide insertions, and short or long deletions in the SBE2 gene, were classified into eight mutant lines. Three mutants, M6, M7 and M8, with long fragment deletions in the second exon of SBE2 showed no accumulation of SBE2 protein. After harvest from the field, significantly higher amylose (up to 56% in apparent amylose content) and resistant starch (up to 35%) was observed in these mutants compared with the wild type, leading to darker blue coloration of starch granules after quick iodine staining and altered starch viscosity with a higher pasting temperature and peak time. Further 1H-NMR analysis revealed a significant reduction in the degree of starch branching, together with fewer short chains (degree of polymerization [DP] 15-25) and more long chains (DP>25 and especially DP>40) of amylopectin, which indicates that cassava SBE2 catalyzes short chain formation during amylopectin biosynthesis. Transition from A- to B-type crystallinity was also detected in the starches. Our study showed that CRISPR/Cas9-mediated mutagenesis of starch biosynthetic genes in cassava is an effective approach for generating novel varieties with valuable starch properties for food and industrial applications.

Relative importance of branching enzyme isoforms in determining starch fine structure and physicochemical properties of indica rice.

KEY MESSAGE: Down-regulation of starch branching enzymes alters fine structure and starch properties, especially the B-type crystalline pattern and extremely high amylose content identified in the BEIIb-deficiency mutant in the indica rice. The relative importance of the starch branching enzymes in determining the molecular fine structure and starch functional properties were uncovered in this study. An indica rice, Guangluai 4 with high amylose content (AC) and high gelatinization temperature (GT) was used to generate the clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein-9 (Cas9) knockout lines. Five mutant lines were identified including be1-1, be1-2, be2a-1, be2a-2 and be2b-1, and analysis of western blot showed the CRISPR/Cas9 system was successful in inducing mutations in the targeted genes. AC of be2b-1 (34.1%) was greater than that of wild type (WT) (27.4%) and other mutants. Mutations of either BEI or BEIIa did not alter the starch crystallite pattern (A-type). The BEIIb deficiency caused an opaque endosperm phenotype, changed the crystallite pattern from A- to B-type, and dramatically increased the degree of ordered structure, the relative proportion of amylose chains and intermediate to long amylopectin chains, average chain length of amylopectin molecules as well as GT. The BEIIa deficiency had no effect on the proportion of amylose chains, the length of amylopectin intermediate-long chains, conclusion temperature and enthalpy of gelatinization. Down-regulation of BEI increased the proportion of shortest amylopectin chains (fa) but decreased the proportion of long amylopectin chains (fb2 and fb3), leading to a lower GT. It is concluded that the relative importance in determining starch fine structures and functionality was in the order of BEIIb > BEI > BEIIa. Our results provide new information for utilizations of BE-deficient mutants in rice quality breeding.

Starch branching enzymes as putative determinants of postharvest quality in horticultural crops.

Starch branching enzymes (SBEs) are key determinants of the structure and amount of the starch in plant organs, and as such, they have the capacity to influence plant growth, developmental, and fitness processes, and in addition, the industrial end-use of starch. However, little is known about the role of SBEs in determining starch structure-function relations in economically important horticultural crops such as fruit and leafy greens, many of which accumulate starch transiently. Further, a full understanding of the biological function of these types of starches is lacking. Because of this gap in knowledge, this minireview aims to provide an overview of SBEs in horticultural crops, to investigate the potential role of starch in determining postharvest quality. A systematic examination of SBE sequences in 43 diverse horticultural species, identified SBE1, 2 and 3 isoforms in all species examined except apple, olive, and Brassicaceae, which lacked SBE1, but had a duplicated SBE2. Among our findings after a comprehensive and critical review of published data, was that as apple, banana, and tomato fruits ripens, the ratio of the highly digestible amylopectin component of starch increases relative to the more digestion-resistant amylose fraction, with parallel increases in SBE2 transcription, fruit sugar content, and decreases in starch. It is tempting to speculate that during the ripening of these fruit when starch degradation occurs, there are rearrangements made to the structure of starch possibly via branching enzymes to increase starch digestibility to sugars. We propose that based on the known action of SBEs, and these observations, SBEs may affect produce quality, and shelf-life directly through starch accumulation, and indirectly, by altering sugar availability. Further studies where SBE activity is fine-tuned in these crops, can enrich our understanding of the role of starch across species and may improve horticulture postharvest quality.

Association among starch storage, metabolism, related genes and growth of Moso bamboo (Phyllostachys heterocycla) shoots.

BACKGROUND: Both underground rhizomes/buds and above-ground Moso bamboo (Phyllostachys heterocycla) shoots/culms/branches are connected together into a close inter-connecting system in which nutrients are transported and shared among each organ. However, the starch storage and utilization mechanisms during bamboo shoot growth remain unclear. This study aimed to reveal in which organs starch was stored, how carbohydrates were transformed among each organ, and how the expression of key genes was regulated during bamboo shoot growth and developmental stages which should lay a foundation for developing new theoretical techniques for bamboo cultivation. RESULTS: Based on changes of the NSC content, starch metabolism-related enzyme activity and gene expression from S0 to S3, we observed that starch grains were mainly elliptical in shape and proliferated through budding and constriction. Content of both soluble sugar and starch in bamboo shoot peaked at S0, in which the former decreased gradually, and the latter initially decreased and then increased as shoots grew. Starch synthesis-related enzymes (AGPase, GBSS and SBE) and starch hydrolase (α-amylase and ß-amylase) activities exhibited the same dynamic change patterns as those of the starch content. From S0 to S3, the activity of starch synthesis-related enzyme and starch amylase in bamboo rhizome was significantly higher than that in bamboo shoot, while the NSC content in rhizomes was obviously lower than that in bamboo shoots. It was revealed by the comparative transcriptome analysis that the expression of starch synthesis-related enzyme-encoding genes were increased at S0, but reduced thereafter, with almost the same dynamic change tendency as the starch content and metabolism-related enzymes, especially during S0 and S1. It was revealed by the gene interaction analysis that AGPase and SBE were core genes for the starch and sucrose metabolism pathway. CONCLUSIONS: Bamboo shoots were the main organ in which starch was stored, while bamboo rhizome should be mainly functioned as a carbohydrate transportation channel and the second carbohydrate sink. Starch metabolism-related genes were expressed at the transcriptional level during underground growth, but at the post-transcriptional level during above-ground growth. It may be possible to enhance edible bamboo shoot quality for an alternative starch source through genetic engineering.

Combined application of biochar and nitrogen fertilizer promotes the activity of starch metabolism enzymes and the expression of related genes in rice in a dual cropping system.

BACKGROUND: Overuse of chemical fertilizer highly influences grain filling rate and quality of rice grain. Biochar is well known for improving plant growth and grain yield under lower chemical fertilization. Therefore field trials were conducted in the early and late seasons of 2019 at Guangxi University, China to investigate the effects of combined biochar (B) and nitrogen (N) application on rice yield and yield components. There were a total of eight treatments: N1B0, 135 kg N ha- 1+ 0 t B ha- 1; N2B0,180 kg N ha- 1+ 0 t B ha- 1; N1B1,135 kg N ha- 1+ 10 t B ha- 1; N1B2,135kg N ha- 1+ 20 t B ha- 1; N1B3,135 kg N ha- 1+ 30 t B ha- 1; N2B1,180 kg N ha- 1+ 10 t B ha- 1; N2B2,180 kg N ha- 1+ 20 t B ha- 1; and N2B3,180 kg N ha- 1+ 30 t B ha- 1. RESULTS: Biochar application at 30 t ha- 1combined with low N application (135 kg ha- 1) increased the activity of starch-metabolizing enzymes (SMEs) during the early and late seasons compared with treatments without biochar. The grain yield, amylose concentration, and starch content of rice were increased in plots treated with 30 t B ha-1and low N. RT-qPCR analysis showed that biochar addition combined with N fertilizer application increased the expression of AGPS2b, SSS1, GBSS1, and GBSE11b, which increased the activity of SMEs during the grain-filling period. CONCLUSION: Our results suggest that the use of 20 to 30 t B ha- 1coupled with 135 kg N ha- 1 is optimal for improving the grain yield and quality of rice.

Cyanobacterial branching enzymes bind to α-glucan via surface binding sites.

Besides their catalysis, specific interactions between starch/glycogen processing enzymes and their substrates have been reported. Multiple branching enzyme (BE) isoforms, BE1, BE2, and BE3, have been found in a limited number of cyanobacterial species that are characterized by amylopectin accumulation. Seven surface binding sites (SBSs) located away from the active site have been identified in crystal structures of cyanobacterial BE1 from Crocosphaera subtropica (Cyanothece sp.) ATCC 51142 (51142BE1). In the present study, binding affinity toward amylopectin, amylose, and glycogen was investigated for wild-type 51142BE1 and its mutants (residues at SBSs important for sugar-binding were replaced by alanine). These enzymes showed retarded mobility during electrophoresis in non-denaturing polyacrylamide gels in the presence of polysaccharides. This was caused by interactions between the enzymes and the polysaccharides, enabling calculation of the dissociation constants (Kd values) of the enzymes toward the polysaccharides. Mutational analysis indicated that particular domains of the protein (domains A and C) were involved in the polysaccharide binding. Kd values toward the polysaccharides were also measured for 10 BE isoforms (five BE1, three BE2, and two BE3) from 5 cyanobacterial strains. All BEs displayed much lower Kd values (higher affinity) toward amylopectin and amylose than toward glycogen, as described for plant BEs. In addition, one BE2 displayed exceptionally high Kd values (low affinity), while two BE3 exhibited multiple Kd values to all polysaccharides. These results could be ascribed to sequence variations in the SBSs, irrespective of the catalytic specificity.

Expression and characterization of 1,4-α-glucan branching enzyme from Microvirga sp. MC18 and its application in the preparation of slowly digestible starch.

Nutraceuticals containing modified starch with increased content of slowly-digestible starch (SDS) may reduce the prevalence of obesity, diabetes and cardiovascular diseases due to its slow digestion rate. Enzymatic methods for the preparation of modified starch have attracted increasing attention because of their low environmental impact, safety and specificity. In this study, the efficient glucan branching enzyme McGBE from Microvirga sp. MC18 was identified, and its relevant properties as well as its potential for industrial starch modification were evaluated. The purified McGBE exhibited the highest specificity for potato starch, with a maximal specific activity of 791.21 U/mg. A time-dependent increase in the content of α-1,6 linkages from 3.0 to 6.0% was observed in McGBE-modified potato starch. The proportion of shorter chains (degree of polymerization, DP < 13) increased from 29.2 to 63.29% after McGBE treatment, accompanied by a reduction of the medium length chains (DP 13-24) from 52.30 to 35.99% and longer chains (DP > 25) from 18.51 to 0.72%. The reduction of the storage modulus (G') and retrogradation enthalpy (ΔHr) of potato starch with increasing treatment time demonstrated that McGBE could inhibit the short- and long-term retrogradation of starch. Under the optimal conditions, the SDS content of McGBE-modified potato starch increased by 65.8% compared to native potato starch. These results suggest that McGBE has great application potential for the preparation of modified starch with higher SDS content that is resistant to retrogradation.

Enzymatic properties of an efficient glucan branching enzyme and its potential application in starch modification.

Glucan branching enzymes (GBEs, EC 2.4.1.18) catalyze the formation of α-1,6-linked branch in starch, which is important for the starch modification with prospective properties. In this study, the aqGBE gene encoding an efficient glucan branching enzyme was cloned from Aquabacterium sp. strain A7-Y and successfully expressed in Escherichia coli BL21 (DE3). The specific activity of the purified recombinant enzyme rAqGBE was 2850 U/mg with potato starch as the optimal substrate, and the Km and Vmax values of rAqGBE were 1.18 mg/mL and 588.2 µmol/min/mg, respectively. Enzymological characterization showed that rAqGBE exhibits its optimal activity under the condition of 40 °C and pH 7.0, respectively, which is independent of calcium ions. Otherwise, rAqGBE-treated potato starch showed different chain length distribution compared with control, the numbers of short chains (degree of polymerization, DP < 7) and long chains (DP > 25) increased from 4.5% to 9.6% and 6.1%-15.7% after enzymatic treatment, respectively. In starch anti-ageing assay, with minimum usage of 0.8 mg rAqGBE per g starch, the rAqGBE-treated potato starch exhibited reduced retrogradation properties. Our results indicate that the branching enzyme AqGBE may therefore be a promising tool for the enzymatic modification of starch.

Identification of Structure-Controlling Rice Biosynthesis Enzymes.

The main enzymes controlling the chain-length distributions (CLDs) of starches are starch synthases (SSs), starch branching enzymes (SBEs), and debranching enzymes (DBEs), which have various isoforms, denoted as SSI, SSII-1, etc. Different isozymes dominate the CLD in different ranges of degrees of polymerization (DPs). Models have been developed for the CLDs in terms of the activities of isoforms of these enzymes, in terms of two parameters: ßi, which is the ratio of the activity of SBE to that of SS in set i, and hi, which is the relative activity of SS in that set. These provide good fits to data but without specifying which isozymes are in set i. Here, CLDs for amylopectin and amylose synthesis in rice endosperm are explored. Molecular weight distributions of the different chains formed in 87 rice varieties were obtained using size-exclusion chromatography following enzymatic debranching (converting a complex branched macromolecule to linear polymers), and fitted by the biosynthesis-based models. The mutants of each isoform among tested rice varieties were identified by amino-acid mutations in coding sequences based on the extraction and analysis of whole gene sequences. The significant differences between mutant groups of different isoforms indicate that SSI, SSII-3, SSIII-1, SSIII-2, and SBEI as well as GBSSI (an isozyme of granule-bound starch synthase) belong to the enzymes sets that control amylose biosynthesis. Further, GBSSI is in the enzyme sets that control amylopectin chains. This enables specification of all isozymes and the DP range, which they dominate, over the entire DP range. As the CLD controls many functional properties of rice, this can help breeders target and develop improved rice species.