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1.
BMC Plant Biol ; 21(1): 477, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34670492

RESUMO

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.


Assuntos
Metabolismo dos Carboidratos/genética , Proteínas de Plantas/metabolismo , Poaceae/genética , Amido/metabolismo , Transcriptoma , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Amilases/genética , Amilases/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/fisiologia , Brotos de Planta/ultraestrutura , Poaceae/crescimento & desenvolvimento , Poaceae/fisiologia , Poaceae/ultraestrutura , Rizoma/genética , Rizoma/crescimento & desenvolvimento , Rizoma/fisiologia , Rizoma/ultraestrutura
2.
BMC Plant Biol ; 21(1): 479, 2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34674662

RESUMO

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.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Produtos Agrícolas/enzimologia , Isoenzimas , Amido/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Motivos de Aminoácidos , Amilopectina/metabolismo , Amilose/metabolismo , Produtos Agrícolas/genética , Produtos Agrícolas/normas , Grão Comestível , Armazenamento de Alimentos , Frutas , Horticultura , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubérculos , Açúcares/metabolismo , Verduras
3.
Enzyme Microb Technol ; 150: 109882, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489035

RESUMO

Glycogen branching enzymes (GBEs; 1,4-α-glucan branching enzyme; E.C. 2.4.1.18) have so far been described to be capable of both α-1,6-transglycosylation (branching) and α-1,4-hydrolytic activity. The aim of the present study was to elucidate the mode of action of three distantly related GBEs from the glycoside hydrolase family 13 by in depth analysis of the activity on a well-defined substrate. For this purpose, the GBEs from R. marinus (RmGBE), P. mobilis (PmGBE1), and B. fibrisolvens (BfGBE) were incubated with a highly pure fraction of a linear substrate of 18 anhydroglucose units. A well-known and characterized branching enzyme from E. coli (EcGBE) was also taken along. Analysis of the chain length distribution over time revealed that, next to hydrolytic and branching activity, all three GBEs were capable of generating chains longer than the substrate, clearly showing α-1,4-transglycosylation activity. Furthermore, the GBEs used those elongated chains for further branching. The sequential activity of elongation and branching enabled the GBEs to modify the substrate to a far larger extent than would have been possible with branching activity alone. Overall, the three GBEs acted ambiguous on the defined substrate. RmGBE appeared to have a strong preference towards transferring chains of nine anhydroglucose units, even during elongation, with a comparably low activity. BfGBE generated an array of elongated chains before using the chains for introducing branches while PmGBE1 exhibited a behaviour intermediate of the other two enzymes. On the basis of the mode of action revealed in this research, an updated model of the mechanism of GBEs was proposed now including the α-1,4-transglycosylation activity.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucanos , Glicogênio , Especificidade por Substrato
4.
Int J Biol Macromol ; 184: 551-557, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34171255

RESUMO

Modified potato starch with slower digestion may aid the development of new starch derivatives with improved nutritional values, and strategies to increase nutritional fractions such as resistant starch (RS) are desired. In this study, a correspondence between starch structure and enzymatic resistance was provided based on the efficient branching enzyme AqGBE, and modified starches with different amylose content (Control, 100%; PS1, 90%; PS2, 72%; PS3, 32%; PS4, 18%) were prepared. Through SEM observation, NMR and X-ray diffraction analyses, we identified that an increased proportion of α-1,6-linked branches in potato starch changes its state of granule into large pieces with crystallinity. Molecular weight and chain-length distribution analysis showed a decrease of molecular weight (from 1.1 × 106 to 1.1 × 105 g/mol) without an obvious change of chain-length distribution in PS1, while PS2-4 exhibited an increased proportion of DP 6-12 with a stable molecular weight distribution, indicating a distinct model of structural modification by AqGBE. The enhancement of peak viscosity was related to increased hydrophobic interactions and pieces state of PS1, while the contents of SDS and RS in PS1 increased by 37.7 and 49.4%, respectively. Our result provides an alternative way to increase the RS content of potato starch by branching modification.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Burkholderiales/enzimologia , Solanum tuberosum/química , Amido/química , Amilose/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Estrutura Molecular , Peso Molecular , Viscosidade , Difração de Raios X
5.
J Agric Food Chem ; 69(20): 5755-5763, 2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-33988022

RESUMO

The 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) catalyzes the formation of α-1,6 branching points in starch and plays a key role in synthesis. To obtain mechanistic insights into the catalytic action of the enzyme, we first determined the crystal structure of GBE from Rhodothermus obamensis STB05 (RoGBE) to a resolution of 2.39 Å (PDB ID: 6JOY). The structure consists of three domains: domain A, domain C, and the carbohydrate-binding module 48 (CBM48). An engineered truncated mutant lacking the CBM48 domain (ΔCBM48) showed significantly reduced ligand binding affinity and enzyme activity. Comparison of the structures of RoGBE with other GBEs showed that CBM48 of RoGBE had a longer flexible loop. Truncation of the flexible loops resulted in reduced binding affinity and activity, thereby substantiating the importance of the optimum loop structure for catalysis. In essence, our study shows that CBM48, especially the flexible loop, plays an important role in substrate binding and enzymatic activity of RoGBE. Further, based on the structural analysis, kinetics, and activity assays on wild type and mutants, as well as homology modeling, we proposed a mechanistic model (called the "lid model") to illustrate how the flexible loop triggers substrate binding, ultimately leading to catalysis.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Rhodothermus , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Glucanos , Rhodothermus/metabolismo , Especificidade por Substrato
6.
Biomacromolecules ; 22(5): 2148-2159, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33914519

RESUMO

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.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Oryza , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Amilopectina , Amilose , Endosperma/genética , Oryza/genética , Amido
7.
Carbohydr Polym ; 262: 117968, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33838833

RESUMO

Enzymatically rearranging α-1,4 and α-1,6 glycosidic bonds in starch is a green approach to regulating its digestibility. A two-step modification process successively catalyzed by 1,4-α-glucan branching enzymes (GBEs) from Rhodothermus obamensi STB05 (Ro-GBE) and Geobacillus thermoglucosidans STB02 (Gt-GBE) was investigated as a strategy to reduce the digestibility of corn starch. This dual GBE modification process caused a reduction of 25.8 % in rapidly digestible starch fraction in corn starch, which were more effective than single GBE-catalyzed modification with the same duration. Structural analysis indicated that the dual GBE modified product contained higher branching density, more abundant short branches, and shorter external chains than those in single GBE-modified product. These results demonstrated that a moderate Ro-GBE treatment prior to starch gelatinization caused several suitable alterations in starch molecules, which promoted the transglycosylation efficiency of the following Gt-GBE treatment. This dual GBE-catalyzed modification process offered an efficient strategy for regulating starch digestibility.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/química , Glicosídeos/química , Amido/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Amilose/química , Amilose/metabolismo , Bacillaceae/enzimologia , Digestão , Glicosídeos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Rhodothermus/enzimologia , Amido/metabolismo
8.
Arch Biochem Biophys ; 702: 108821, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33662318

RESUMO

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.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Cianobactérias/enzimologia , Glucanos/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Sequência de Aminoácidos , Domínio Catalítico , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Alinhamento de Sequência
9.
Int J Biol Macromol ; 174: 110-119, 2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33515569

RESUMO

To obtain porous starch granules with higher absorption capacities, three types of enzyme combinations were adopted to modify wheat and maize starches: (1) sequential α-amylase (AA) â†’ glucoamylase (GA); (2) sequential branching enzyme (BE) â†’ GA; and (3) sequential AA→BE→GA. The results indicated that AA→BE→GA treatment had a most optimal influence on porous starches. Compared to AA→GA and BE→GA, the mesopores in wheat starch granules treated with AA→BE→GA decreased by 52.82 and 48.70%, respectively. Conversely, the macropores increased by 216.68 and 138.18%, respectively. While for maize starch, the percentages of mesopores and macropores hardly changed after three enzyme combinations. Comparing the three enzyme treatments showed that pore volume (0.005 and 0.007 cm3/g) and pore size (36.35 and 26.54 nm) were largest in the AA→BE→GA treated wheat and maize starches, respectively. Compared to the AA→GA and BE→GA, the adsorption capacities for oil, dye and heavy metal ions, wheat starch treated with AA→BE→GA increased by 46.61 and 242.33%, and 44.52 and 134.41%, and 28.83 and 271.72%, respectively. Correspondingly, that of maize starch increased by 29.71 and 133.29%, and 42.92 and 79.93%, and 28.16 and 161.43%, respectively. These results may provide a new and valuable enzyme combination for optimising porous starch granules with higher absorption capacities.


Assuntos
Adsorção/fisiologia , Amido/química , Triticum/química , Zea mays/química , Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Amilases/química , Amilose/química , Glucana 1,4-alfa-Glucosidase/química , Glucana 1,4-alfa-Glucosidase/metabolismo , Hidrólise , Porosidade , Temperatura , Água/química , alfa-Amilases/química , alfa-Amilases/metabolismo
10.
Food Chem ; 343: 128485, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33172750

RESUMO

The aim of this study was to prepare a supporting carrier, namely highly branched corn starch (HBCS), and to investigate its encapsulation property with ascorbic acid (AA). High amylose corn starch was converted into HBCS via dual enzymatic modification by successively using α-amylase and glycogen branching enzyme. The results showed that the ratio of α-1, 6 linkage of HBCS increased by 1.93%, and a short-to-medium chain length distribution with a compact branched conformation was formed, which suggested HBCS could be a potential highly branched carrier. The HBCS-AA inclusion complex was formed as confirmed by differential scanning calorimetry. The release of AA conformed to the pseudo-Fickian diffusion mechanism and followed the first-order kinetics. Meanwhile, the photostability and thermostability of the embedded AA were moderately enhanced. These findings suggest that HBCS provides new insights into the preparation of wall materials and can be potentially used to deliver AA into food systems.


Assuntos
Ácido Ascórbico/química , Amido/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Cápsulas , alfa-Amilases/metabolismo
11.
Protein Expr Purif ; 178: 105779, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33115653

RESUMO

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.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Proteínas de Bactérias , Burkholderiales/genética , Amido/química , Enzima Ramificadora de 1,4-alfa-Glucana/biossíntese , Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enzima Ramificadora de 1,4-alfa-Glucana/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Burkholderiales/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
12.
J Agric Food Chem ; 68(47): 13791-13797, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33166453

RESUMO

Disulfide bonds play crucial roles in thermostabilization, recognition, or activation of proteins. They are vital in maintaining the respective conformations of globular structures, thereby enhancing thermostability. Bioinformatic approaches provide practical strategies to build disulfide bonds based on structural information. We constructed nine mutants by rational analysis of the 1,4-α-glucan branching enzyme (EC 2.4.1.18) from Geobacillus thermoglucosidans STB02, which catalyzes the synthesis of α-1,6-glucosidic bonds by acting on α-(1,4) and/or α-(1,6) glucosidic linkages. Four of the mutations enhanced thermostability, and five of them had adverse or negligible effects on stability. Circular dichroism spectra and intrinsic fluorescence analysis showed that introducing disulfide bonds might only affect secondary structures. The results also demonstrated that the distances of Cα carbons and thiol groups, as well as the sequence between the two cysteines, need to be considered when designing disulfide bonds.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Geobacillus , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Bacillaceae , Dissulfetos , Estabilidade Enzimática , Geobacillus/genética , Glucanos
13.
Proc Natl Acad Sci U S A ; 117(42): 26503-26512, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33020297

RESUMO

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.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Oryza/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/química , Amilopectina/biossíntese , Amilopectina/química , Amilose/biossíntese , Amilose/química , Metabolismo dos Carboidratos , Grão Comestível/genética , Endosperma/metabolismo , Mutação , Oryza/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Sementes/metabolismo , Amido/biossíntese , Sintase do Amido/química , Sintase do Amido/genética , Sintase do Amido/metabolismo
14.
Genes Genomics ; 42(11): 1227-1238, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32901332

RESUMO

BACKGROUND: The digestibility of starch is important for the nutritive value of staple food. Although several genes are responsible for resistant starch (RS) and slowly digestible starch (SDS), gaps persist concerning the molecular basis of RS and SDS formation due to the complex genetic mechanisms of starch digestibility. OBJECTIVES: The objective of this study was to identify new genes for starch digestibility in rice and interprete the genetic mechanisms of RS and SDS by GWAS. METHODS: Genome-wide association studies were conducted by associating the RS and SDS phenotypes of 104 re-sequenced rice lines to an SNP dataset of 2,288,867 sites using a compressed mixed linear model. Candidate genes were identified according to the position of the SNPs based on data from the MSU Rice Genome Annotation Project. RESULTS: Seven quantitative trait loci (QTLs) were detected to be associated with the RS content, among which the SNP 6 m1765761 was located on Waxy. Starch branching enzymes IIa (BEIIa) close to QTL qRS-I4 was detected and further identified as a specific candidate gene for RS in INDICA. Two QTLs were associated with SDS, and the LOC_Os09g09360 encoding lipase was identified as a causal gene for SDS. CONCLUSIONS: GWAS is a valid strategy to genetically dissect the formation of starch digestion properties in rice. RS formation in grains is dependent on the rice type; lipid might also contribute to starch digestibility and should be an alternative factor to improve rice starch digestibility.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Lipase/genética , Oryza/genética , Amido Resistente , Amido/genética , Digestão/genética , Genoma de Planta/genética , Estudo de Associação Genômica Ampla , Humanos , Oryza/química , Oryza/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Amido/metabolismo
15.
Placenta ; 99: 131-140, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32798765

RESUMO

INTRODUCTION: The placenta performs a range of functions to support fetal growth. In addition to facilitating nutrient transport, the placenta also stores glucose as glycogen, which is thought to maintain fetal glucose supply during late gestation. However, evidence to support such a role is currently lacking. Similarly, our understanding of the dynamics of placental glycogen metabolism in normal mouse pregnancy is limited. METHODS: We quantified the placental glycogen content of wild type C57BL/6JOlaHsd mouse placentas from mid (E12.5) to late (E18.5) gestation, alongside characterising the temporal expression pattern of genes encoding glycogenesis and glycogenolysis pathway enzymes. To assess the potential of the placenta to produce glucose, we investigated the spatiotemporal expression of glucose 6-phosphatase by qPCR and in situ hybridisation. Separate analyses were undertaken for placentas of male and female conceptuses to account for potential sexual dimorphism. RESULTS: Placental glycogen stores peak at E15.5, having increased over 5-fold from E12.5, before declining by a similar extent by E18.5. Glycogen stores were 17% higher in male placentas than in females at E15.5. Expression of glycogen branching enzyme (Gbe1) was reduced ~40% towards term. Expression of the glucose 6-phosphatase isoform G6pc3 was enriched in glycogen trophoblast cells and increased towards term. DISCUSSION: Reduced expression of Gbe1 suggests a decline in glycogen branching towards term. Expression of G6pc3 by glycogen trophoblasts is consistent with an ability to produce and release glucose from glycogen stores. However, the ultimate destination of the glucose generated from placental glycogen remains to be elucidated.


Assuntos
Glicogênio/metabolismo , Placenta/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Gravidez , Trofoblastos/metabolismo
16.
Carbohydr Polym ; 247: 116681, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32829809

RESUMO

Two high amylose (HAM) inbred lines with apparent amylose contents of 55 % and 62 %, respectively, were selected to explore the relationship between molecular structure and gene expression of starch-synthase involved enzymes. GPC analysis of debranched starches showed that the HAM starches (HAMSs) had shorter amylose chains and longer amylopectin chains than normal maize starch (NMS). FACE analysis showed that these HAMSs had a higher content of amylopectin chains of DP > 21. Quantitative Real-Time PCR analysis showed that the HAM lines had specifically low expression of the starch branching enzyme IIb (SBEIIb), and the starch synthase IIIa (SSIIIa) homologue, and high expression of the isoamylase 2 (ISA2), potentially suppressing the generation of amylopectin molecules through deficient branching and excessive debranching process, thereby increasing the relative amylose content. A high expression of GBSS1 was potentially associated with increased short amylose chain lengths in HAMSs.


Assuntos
Amilose/química , Sintase do Amido/genética , Sintase do Amido/metabolismo , Amido/biossíntese , Amido/química , Zea mays/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Amilopectina/análise , Amilopectina/química , Metabolismo dos Carboidratos , Cromatografia em Gel , Eletroforese/métodos , Isoamilase/metabolismo , Estrutura Molecular , Amido/análise , Zea mays/metabolismo
17.
Carbohydr Polym ; 247: 116729, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32829851

RESUMO

Twenty-four branched maltodextrins were synthesized from eight starches using three thermostable microbial glycogen branching enzymes. The maltodextrins have a degree of branching (DB) ranging from 5 % to 13 %. This range of products allows us to explore the effect of DB on the digestibility, which was quantified under conditions that mimic the digestion process in the small intestine. The rate and extent of digestibility were analyzed using the logarithm of the slope method, revealing that the branched maltodextrins consist of a rapidly and slowly digestible fraction. The amount of slowly digestible maltodextrin increases with an increasing DB. Surprisingly, above 10 % branching the fraction of slowly digestible maltodextrin remains constant. Nevertheless, the rate of digestion of the slowly digestible fraction was found to decline with increasing DB and shorter average internal chain length. These observations increase the understanding of the structural factors important for the digestion rate of branched maltodextrins.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Bactérias/enzimologia , Gelatina/química , Glucana 1,4-alfa-Glucosidase/metabolismo , Glicogênio/metabolismo , alfa-Amilases Pancreáticas/metabolismo , Amido/química , Digestão , Hidrólise , Cinética
18.
Theor Appl Genet ; 133(10): 2961-2974, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32651668

RESUMO

KEY MESSAGE: Catalytically active indica SSIIa allele in high amylose rice with down-regulated japonica SBEIIb can increase starch content and modify the starch structure and properties without changing its amylose content. Rice (Oryza sativa) genotypes with inactive starch synthase IIa (SSIIa) with recessive variants of starch branching enzyme IIb (SBEIIb) exhibit a range of alterations in grain phenotype, starch granule morphology, starch granule bound proteins, starch structure, and functional properties. However, the interactions between the two enzymes have not been thoroughly investigated yet. We analysed recombinant rice lines having down-regulated SBEIIb expression (SBEIIbDR) with either indica or japonica type SSIIa (SSIIaind or SSIIajap). In SBEIIbDR rice starch granules, the increased abundance of two protein bands (SSI and SSIIa) was found with eight additional protein bands not generally associated with starch granules. The amount of SSIIa was higher in SSIIaindSBEIIbDR than SSIIajapSBEIIbDR, which indicated that indica type SSIIa, possibly in the monomer form, was extensively involved in starch biosynthesis in the SBEIIbDR endosperm. Furthermore, SSIIaindSBEIIbDR grains had higher total starch content and higher starch swelling power than SSIIajapSBEIIbDR lines, but the amylopectin gelatinization temperatures and enthalpy and the apparent amylose content remained similar. In summary, this work suggests that SSIIaind can partly compensate for the alteration of starch synthesis resulting from the SBEIIb down-regulation in japonica background without reducing its amylose content. The study provides insight into the starch structural and textural improvements of high amylose starch.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Amilose/química , Oryza/genética , Proteínas de Plantas/genética , Sintase do Amido/genética , Amido/química , Alelos , Cruzamentos Genéticos , Regulação para Baixo , Grão Comestível/genética , Endosperma/química , Regulação da Expressão Gênica de Plantas , Genótipo , Oryza/enzimologia , Plantas Geneticamente Modificadas/enzimologia
19.
Curr Opin Plant Biol ; 55: 109-117, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32428846

RESUMO

The traditional view of starch metabolism has focused on the multiplicity of enzymes and enzyme isoforms contributing to the production of the constituent polymers, amylopectin and amylose. However, knowledge of these enzymes has not provided a full insight into many aspects of starch biosynthesis. This enzyme-centered view has recently been augmented by the discovery and characterization of novel proteins with proposed regulatory, scaffolding, and interactive roles. This begins to reveal an unprecedented level of complexity beyond mere glucan biosynthesis, enabling us to envisage how starch granules are initiated and grow into specific forms, allowing it to serve biological roles beyond just carbohydrate storage. This review focuses on very recent findings in this vibrant field, highlighting the evolutionary novelty.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Amilopectina , Amilose , Evolução Biológica , Metabolismo dos Carboidratos , Amido
20.
Pediatr Int ; 62(10): 1145-1150, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32374048

RESUMO

BACKGROUND: Glycogen storage diseases (GSD) are disorders of autosomal recessive carbohydrate metabolism, characterized by glycogen accumulation. The liver and muscle tissue are commonly affected but patients may present with different clinical manifestations. The presence of glycogen can be demonstrated in biopsies and definitive diagnosis can be made by enzymatic or molecular analysis. The aim of this study was to determine specific gene mutations in our cases with GSD. METHODS: Thirty-eight patients with clinical and laboratory diagnoses of GSD were studied. Thirty-two patients had undergone genetic analysis. In our study, a next-generation sequencing panel was used. RESULTS: Five novel variants of uncertain significance (VUS), which were likely to be pathogenic, were detected in seven patients. Two new pathogenic variations of c.927delT (p.Phe309LeufsTer4) homozygous and c.44C>G (p.Ser15Ter) homozygous in the G6PC gene were detected in two GSD type Ia patients. In our two non-sibling GSD type III patients, c.1439T>G (p.Leu480Arg) homozygous novel-VUS was detected in the AGL gene. In our GSD type IV patient, c.1054G>C (p.Asp352His) homozygous novel-VUS was detected in the GBE1 gene. In GSD type VI, two sibling patients had a c.1454A>G (p.Asn485Ser) homozygous novel-VUS change in the PYGL gene. CONCLUSIONS: We determined the gene mutations specific to cohorts in our cases with GSD. The novel pathogenic, likely pathogenic, and VUS changes identified will contribute to the relationship between the patients' clinical and laboratory findings.


Assuntos
Doença de Depósito de Glicogênio/genética , Mutação , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Metabolismo dos Carboidratos/genética , Criança , Análise Mutacional de DNA , Feminino , Sistema da Enzima Desramificadora do Glicogênio/genética , Glicogênio Fosforilase/genética , Doença de Depósito de Glicogênio/patologia , Doença de Depósito de Glicogênio Tipo I/genética , Doença de Depósito de Glicogênio Tipo III/genética , Homozigoto , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Turquia
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