Creation of rice doubled haploids with low amylose content using in vitro anther culture.

In vitro androgenesis is a unique model for producing homozygous doubled haploid plants. The use of haploid biotechnology accelerates to obtain of doubled haploid plants, which is very important in rice breeding. The purpose of this work is to improve the production of doubled haploids in rice anther culture in vitro and selection of doubled haploid plants with valuable traits. The study the influence of nutrient media on the production of calli and plant regeneration processes in anther culture of 35 rice genotypes was revealed a significant influence of nutrient media on callus production. It was shown that the addition to culture medium phytohormones ratio with high level of cytokinin (5.0 mg/L BAP) and a low level of auxin (0.5 mg/L NAA), supplemented with amino acid composition promotes high production of green regenerated plants (68.75%) compared to albino plants (31.25%). As a result, doubled haploid lines of the glutinous variety Violetta were selected, which characterized by a low amylose content variation (from 1.86 to 2.80%). These doubled haploids are superior to the original variety in some yield traits and represent valuable breeding material.

Environment affects starch composition and kernel hardness in temperate maize.

BACKGROUND: Protein percentage and kernel weight affect the endosperm hardness of maize grains. However, changes in starch composition could also modify kernel hardness, which is often predicted through milling ratio. The objective of this work was to evaluate the relationship between changes in starch composition and endosperm hardness, and to assess the effects of protein content and kernel weight on that relationship. For this, we used information obtained from three temperate hybrids in multi-environmental experiments, as well as experiments conducted under controlled conditions designed to modify starch composition. Milling ratio was determined as maize kernel hardness predictor in both experiments and it was correlated with grain weight and grain composition. RESULTS: In both experiments, milling ratio presented a significant Spearman correlation coefficient with amylose/starch ratio. Milling ratio was significantly related to the amylose/starch ratio using a simple fit with datasets from different experiments and hybrids. Increases in amylose/starch ratio were associated with increases in milling ratio. CONCLUSION: Starch composition was related to milling ratio variations among hybrids of different aptitude for dry milling through different environments, regardless of protein content. Thus, increases in the amylose/starch ratio were related to increases in the milling ratio. © 2022 Society of Chemical Industry.

CBM20CP, a novel functional protein of starch metabolism in green algae.

Ostreococcus tauri is a picoalga that contains a small and compact genome, which resembles that of higher plants in the multiplicity of enzymes involved in starch synthesis (ADP-glucose pyrophosphorylase, ADPGlc PPase; granule bound starch synthase, GBSS; starch synthases, SSI, SSII, SSIII; and starch branching enzyme, SBE, between others), except starch synthase IV (SSIV). Although its genome is fully sequenced, there are still many genes and proteins to which no function was assigned. Here, we identify the OT_ostta06g01880 gene that encodes CBM20CP, a plastidial protein which contains a central carbohydrate binding domain of the CBM20 family, and a coiled coil domain at the C-terminus that lacks catalytic activity. We demonstrate that CBM20CP has the ability to bind starch, amylose and amylopectin with different affinities. Furthermore, this protein interacts with OsttaSSIII-B, increasing its binding to starch granules, its catalytic efficiency and promoting granule growth. The results allow us to postulate a functional role for CBM20CP in starch metabolism in green algae. KEY MESSAGE: CBM20CP, a plastidial protein that has a modular structure but lacks catalytic activity, regulates the synthesis of starch in Ostreococcus tauri.

The Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) Has Digestive Capacity to Degrade Complex Substrates: Functional Characterization and Heterologous Expression of an α-Amylase.

Dendroctonus-bark beetles are natural agents contributing to vital processes in coniferous forests, such as regeneration, succession, and material recycling, as they colonize and kill damaged, stressed, or old pine trees. These beetles spend most of their life cycle under stem and roots bark where they breed, develop, and feed on phloem. This tissue is rich in essential nutrients and complex molecules such as starch, cellulose, hemicellulose, and lignin, which apparently are not available for these beetles. We evaluated the digestive capacity of Dendroctonusrhizophagus to hydrolyze starch. Our aim was to identify α-amylases and characterize them both molecularly and biochemically. The findings showed that D. rhizophagus has an α-amylase gene (AmyDr) with a single isoform, and ORF of 1452 bp encoding a 483-amino acid protein (53.15 kDa) with a predicted signal peptide of 16 amino acids. AmyDr has a mutation in the chlorine-binding site, present in other phytophagous insects and in a marine bacterium. Docking analysis showed that AmyDr presents a higher binding affinity to amylopectin compared to amylose, and an affinity binding equally stable to calcium, chlorine, and nitrate ions. AmyDr native protein showed amylolytic activity in the head-pronotum and gut, and its recombinant protein, a polypeptide of ~53 kDa, showed conformational stability, and its activity is maintained both in the presence and absence of chlorine and nitrate ions. The AmyDr gene showed a differential expression significantly higher in the gut than the head-pronotum, indicating that starch hydrolysis occurs mainly in the midgut. An overview of the AmyDr gene expression suggests that the amylolytic activity is regulated through the developmental stages of this bark beetle and associated with starch availability in the host tree.

Purification and functional properties of a novel glucoamylase activated by manganese and lead produced by Aspergillus japonicus.

Microbial amylases are used to produce ethanol, glucose and can be applied in textiles products, detergents and other industries. This study aimed to determine the best carbon source concentration to induce the amylase production by A. japonicus, and its purification and biochemical characterization. For that, this fungus was cultivated in Khanna medium, pH 5.5, for 4 days, at 25°C, in static condition, supplemented with potato starch and maltose in different concentrations. The fungal crude enzymatic extract was purified in a unique elution in DEAE-cellulose column and the molecular mass was determined as 72kDa. The optimum temperature and pH was 65°C and 5.0, respectively. Amylase remained 75% of its activity after one hour at 50°C and was stable in the pH range 3.0-7.0. The analysis of the end-products by thin layer chromatography showed only glucose formation, which characterizes the purified enzyme as a glucoamylase. Amylopectin was the best substrate for the enzyme assay and Mn+2 and Pb+2 were good glucoamylase activators. This activation, in addition to the biochemical characteristics are important results for future biotechnological applications of this glucoamylase in the recycling and deinking process by the paper industries.

Physicochemical properties of nixtamalized corn flours with and without germ.

This research studied the influence of the germ components on the physicochemical properties of cooked corn and nixtamalized corn flours as a function of the calcium hydroxide content (from 0 to 2.1 w/w) and steeping time (between 0 and 9h). A linear relationship was found between calcium content in germ and steeping time used during nixtamalization process. X-ray diffraction analysis showed that calcium carbonate is formed into the germ structure to 2.1 w/w of calcium hydroxide and 9h steeping time. The presence of the germ improves the development of peak viscosity in flours, and it is related to the increases in calcium concentration in germ and the formation of amylose-lipid complexes. No significant changes were observed in palmitic, stearic, oleic and linoleic acids of corn oil. The levels of further corn oil deterioration were 2.1 w/w of calcium hydroxide concentration and 9h of steeping time.

Functional demonstrations of starch binding domains present in Ostreococcus tauri starch synthases isoforms.

BACKGROUND: Starch-binding domains are key modules present in several enzymes involved in polysaccharide metabolism. These non-catalytic modules have already been described as essential for starch-binding and the catalytic activity of starch synthase III from the higher plant Arabidopsis thaliana. In Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, there are three SSIII isoforms, known as Ostta SSIII-A, SSIII-B and SSIII-C. RESULTS: In this work, using in silico and in vitro characterization techniques, we have demonstrated that Ostta SSIII-A, SSIII-B and SSIII-C contain two, three and no starch-binding domains, respectively. Additionally, our phylogenetic analysis has indicated that OsttaSSIII-B, presenting three N-terminal SBDs, is the isoform more closely related to higher plant SSIII. Furthermore, the sequence alignment and homology modeling data gathered showed that both the main 3-D structures of all the modeled domains obtained and the main amino acid residues implicated in starch binding are well conserved in O. tauri SSIII starch-binding domains. In addition, adsorption assays showed that OsttaSSIII-A D2 and SSIII-B D2 domains are the two that make the greatest contribution to amylose and amylopectin binding, while OsttaSSIII-B D1 is also important for starch binding. CONCLUSIONS: The results presented here suggest that differences between OsttaSSIII-A and SSIII-B SBDs in the number of and binding of amino acid residues may produce differential affinities for each isoform to polysaccharides. Increasing the knowledge about SBDs may lead to their employment in biomedical and industrial applications.

Fungi Isolated from Maize (Zea mays L.) Grains and Production of Associated Enzyme Activities.

Filamentous fungi produce a great variety of enzymes, and research on their biotechnological potential has recently intensified. The objective of this work was to identify, at the species level, using DNA barcoding, 46 fungal isolates obtained from maize grains with rot symptoms. We also analyzed the production of extracellular amylases, cellulases, proteases and lipases of 33 of those fungal isolates. The enzymatic activities were evaluated by the formation of a clear halo or a white precipitate around the colonies in defined substrate media. The found fungi belong to the genera Talaromyces, Stenocarpella, Penicillium, Phlebiopsis, Cladosporium, Hyphopichia, Epicoccum, Trichoderma, Aspergillus, Irpex, Fusarium, Microdochium, Mucor and Sarocladium. In the genus Fusarium, the species Fusarium verticillioides was predominant and this genus presented the highest diversity, followed by the genera Aspergillus. The best genera for lipase production were Cladosporium and Penicillium; while Cladosporium, Aspergillus and Penicillium were best for cellulase activity; Hyphopichia, Aspergillus and Irpex for amylase activity; and Cladosporium and Sarocladium for proteases activity. In conclusion, a collection of fungi from maize seeds presenting rotten symptoms were obtained, among which exist important producers of hydrolases.

Whole genome sequencing of elite rice cultivars as a comprehensive information resource for marker assisted selection.

Current advances in sequencing technologies and bioinformatics revealed the genomic background of rice, a staple food for the poor people, and provided the basis to develop large genomic variation databases for thousands of cultivars. Proper analysis of this massive resource is expected to give novel insights into the structure, function, and evolution of the rice genome, and to aid the development of rice varieties through marker assisted selection or genomic selection. In this work we present sequencing and bioinformatics analyses of 104 rice varieties belonging to the major subspecies of Oryza sativa. We identified repetitive elements and recurrent copy number variation covering about 200 Mbp of the rice genome. Genotyping of over 18 million polymorphic locations within O. sativa allowed us to reconstruct the individual haplotype patterns shaping the genomic background of elite varieties used by farmers throughout the Americas. Based on a reconstruction of the alleles for the gene GBSSI, we could identify novel genetic markers for selection of varieties with high amylose content. We expect that both the analysis methods and the genomic information described here would be of great use for the rice research community and for other groups carrying on similar sequencing efforts in other crops.

Genetic parameters and prediction of genotypic values for root quality traits in cassava using REML/BLUP.

The aim of this study was to estimate the genetic parameters and predict the genotypic values of root quality traits in cassava (Manihot esculenta Crantz) using restricted maximum likelihood (REML) and best linear unbiased prediction (BLUP). A total of 471 cassava accessions were evaluated over two years of cultivation. The evaluated traits included amylose content (AML), root dry matter (DMC), cyanogenic compounds (CyC), and starch yield (StYi). Estimates of the individual broad-sense heritability of AML were low (hg(2) = 0.07 ± 0.02), medium for StYi and DMC, and high for CyC. The heritability of AML was substantially improved based on mean of accessions (hm(2) = 0.28), indicating that some strategies such as increasing the number of repetitions can be used to increase the selective efficiency. In general, the observed genotypic values were very close to the predicted average of the improved population, most likely due to the high accuracy (>0.90), especially for DMC, CyC, and StYi. Gains via selection of the 30 best genotypes for each trait were 4.8 and 3.2% for an increase and decrease for AML, respectively, an increase of 10.75 and 74.62% for DMC for StYi, respectively, and a decrease of 89.60% for CyC in relation to the overall mean of the genotypic values. Genotypic correlations between the quality traits of the cassava roots collected were generally favorable, although they were low in magnitude. The REML/BLUP method was adequate for estimating genetic parameters and predicting the genotypic values, making it useful for cassava breeding.

Analysis of genetic diversity and trait correlations among Korean landrace rice (Oryza sativa L.).

This study analyzed 394 Korean rice landrace accessions, including 93 waxy varieties, for polymorphisms using 29 simple sequence repeat (SSR) markers. In total, 381 alleles served as raw data for estimating the genetic diversity (GD) and population structure. The number of alleles per locus ranged from 3 to 44 (average = 13.14). The expected heterozygosity and polymorphism information content (PIC) ranged from 0.0341 to 0.9358 (mean = 0.5623) and from 0.0783 to 0.9367 (mean = 0.5839), respectively. The mean GDs in waxy, low amylose content, intermediate amylose content, and high amylose content (HAC) varieties were 0.6014, 0.5922, 0.5858, and 0.7232, respectively, whereas the mean PIC values for each SSR locus were 0.5701, 0.5594, 0.5550, and 0.6926, respectively. HAC varieties had the highest GD and PIC. Consistent with clustering by genetic distances, a model-based structural analysis revealed 3 subpopulations. Analysis of molecular variance revealed that the between-population component of genetic variance was 22.35%, and that of the within-population component was 77.65%. Significant correlations were observed between eating quality and protein content (r = -0.262), K(+) (r = -0.655), Mg(2+) (r = -0.680), 1000-GW (r = 0.159), and amylose content (r = -0.134). The overall FST value was 0.2235, indicating moderate differentiation among the groups. Analysis of variance of the 3 genetic groups (mean of 9 phenotypic and 5 physicochemical traits) by the Duncan multiple range test showed significant differences in 10 traits. This preliminary study represents a first step toward more efficient conservation and greater utilization of rice landraces to broaden the genetic bases of commercially grown varieties.

Purification, partial characterization, and covalent immobilization-stabilization of an extracellular α-amylase from Aspergillus niveus.

An extracellular amylase secreted by Aspergillus niveus was purified using DEAE fractogel ion exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5 % polyacrylamide gel electrophoresis (PAGE) and 10 % sodium dodecyl sulfate (SDS-PAGE). The enzyme exhibited 4.5 % carbohydrate content, 6.6 isoelectric point, and 60 and 52 kDa molar mass estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The amylase efficiently hydrolyzed glycogen, amylose, and amylopectin. The end-products formed after 24 h of starch hydrolysis, analyzed by thin layer chromatography, were maltose, maltotriose, maltotetraose, and maltopentaose, which classified the studied amylase as an α-amylase. Thermal stability of the α-amylase was improved by covalent immobilization on glyoxyl agarose (half-life of 169 min, at 70 °C). On the other hand, the free α-amylase showed a half-life of 20 min at the same temperature. The optima of pH and temperature were 6.0 and 65 °C for both free and immobilized forms.

RNA interference-mediated silencing of the starch branching enzyme gene improves amylose content in rice.

Amylose and amylopectin are the 2 major components of plant storage starch. The rice starch branching enzyme (RBE) plays an important role in the starch components of rice. In the present study, we selected a specific 195-bp segment from the RBE3 gene to construct hairpin DNA, which was driven by an endosperm-specific high molecular weight glutenin promoter to regulate the biosynthesis of starch. An RNA interference plasmid for the RBE3 gene was constructed to form double-stranded RNA. Following Agrobacterium-mediated rice transformation (in the cultivar Zhonghua 11), 41 transgenic plants were identified using PCR and Southern blot analysis. Semi-quantitative real-time PCR revealed that RBE3 gene expression was significantly reduced in immature transgenic seeds. Transgenic rice amylose content had an average increase of 140%. The highest rice amylose content was 47.61% and the growth rate increased 238% compared to the non-transgenic controls. Branching enzyme II activity was notably reduced, and ADP-glucose pyrophosphorylase, soluble starch synthase, isoamylase, and pullulanase enzyme activity was markedly reduced in T3 seeds. Relative enzyme activity change explained the reduction in thousand-grain weight in transgenic plants. The present study indicated that amylose content was negatively correlated with branching enzyme II activity, spike size, and thousand-grain weight.

Starch determination, amylose content and susceptibility to in vitro amylolysis in flours from the roots of 25 cassava varieties.

BACKGROUND: Cassava cultivars are classified following different criteria, such as cyanogenic glucoside content or starch content. Here, flours from the roots of 25 cassava varieties cultivated simultaneously in a single plantation, were characterized in terms of starch content (SC), amylose content (AC), α-amylolysis index (AI) and gel formation ability. Resistant starch content (RS) was measured in 10 of the samples. RESULTS: Cassava flours exhibited high SC, low AC and low AI values, with differences among varieties. Cluster analysis based on these parameters divided the cultivars in four groups differing mainly in SC and AC. AI and AC were inversely correlated (r = -0.59, P < 0.05) in 18 of the cultivars, suggesting AC as an important factor governing the susceptibility to enzymatic hydrolysis of starch in raw cassava. Differences in susceptibility to amylolysis, assessed by RS, were also recorded in the sample subset analyzed. Most flours yielded pastes or gels upon heating and cooling. Gels differed in their subjective grade of firmness, but none exhibited syneresis, confirming the low retrogradation proclivity of cassava starch. CONCLUSION: Some differences were found among cassava samples, which may be ascribed to inter-cultivar variation. This information may have application in further agronomic studies or for developing industrial uses for this crop.

[The effect of parboiled rice on glycemia in Wistar rats]. / Arroz parboilizado efeito na glicemia de ratos Wistar.

Starch is an important energy source and can represent more than 60% of the calories of the human diet. The starch fraction resistant to enzymatic digestion is called resistant starch. When rice is parboiled, the starch retrogrades with the formation of type 3-resistant starch (retrograded), which presents beneficial effects on the health, since it acts as a prebiotic. In the present study three types of rice were selected, with high, medium and low amylose contents, with the objective of evaluating the effects of conventional and parboiled rice on glycemia in Wistar rats. The samples with high and medium amylose contents were soaked for 6 h at 65 degrees C, and the low amylose sample for 7 h at 70 degrees C. The samples were subsequently autoclaved for 10 minutes at 0.7kgf x cm(-2). Six male Wistar rats were used for each treatment. Seven experimental diets were elaborated, formulated according to AIN-93M, the control diet and diets substituting the carbohydrate source with conventional or parboiled rice. Resistant starch was determined in the diets and glycemia monitored using glucose paper strips, the sample being blood obtained from the distal part of the rat's tail. For the glycemic curve, glycemia was measured in the fasting state and during 90 minutes post-prandial. The results indicated there were no significant differences between the diets formulated with high, medium and low amylose, parboiled or conventionally prepared, with respect to fasting or post-prandial glycemia in Wistar rats.

Arroz parboilizado efeito na glicemia de ratos Wistar

O amido é uma importante fonte energética podendo representar mais de 60% da ingestão calórica na dieta humana. A fração do amido, que é resistente à digestão enzimática, é denominada de amido resistente. Na parboilização de arroz ocorre a retrogradação do amido, com a formação do amido resistente tipo 3 (retrogradado), que apresenta efeitos benéficos à saúde, por atuar como prébiótico. Neste estudo, três amostras de arroz com alta, média e baixa amilose foram selecionadas, com o objetivo de avaliar o efeito de arroz convencional e parboilizado na glicemia de ratos Wistar. As amostras com alta e média amilose foram encharcadas durante 6h a 65°C e a amostra com baixa amilose durante 7h a 70°C, posteriormente foram autoclavadas em pressão 0,7 kgf.cm-2 durante 10 minutos. Utilizouse 6 ratos Wistar, machos, adultos por tratamento. Foram elaboradas 7 dietas experimentais, formuladas de acordo com AIN-93M, dieta controle e, dietas com substituição da fonte de carboidratos por arroz convencional e parboilizado. Determinou-se amido resistente nas dietas e a glicemia foi monitorada por leitura de glicofita, com sangue da parte distal da cauda do rato. Para a curva glicêmica as medidas de glicemia foram feitas em jejum, e ao longo de 90 minutos. Os resultados indicam que não foram encontradas diferenças significativas entre as dietas formuladas com arroz de alta, média e baixa amilose, parboilizado ou convencional na glicemia de jejum e pós-prandial de ratos Wistar.

The effect of parboiled rice on glycemia in Wistar rats. Starch is an important energy source and can represent more than 60% of the calories of the human diet. The starch fraction resistant to enzymatic digestion is called resistant starch. When rice is parboiled, the starch retrogrades with the formation of type 3- resistant starch (retrograded), which presents beneficial effects on the health, since it acts as a prebiotic. In the present study three types of rice were selected, with high, medium and low amylose contents, with the objective of evaluating the effects of conventional and parboiled rice on glycemia in Wistar rats. The samples with high and medium amylose contents were soaked for 6 h at 65ºC, and the low amylose sample for 7 h at 70ºC. The samples were subsequently autoclaved for 10 minutes at 0.7kgf.cm-2. Six male Wistar rats were used for each treatment. Seven experimental diets were elaborated, formulated according to AIN-93M, the control diet and diets substituting the carbohydrate source with conventional or parboiled rice. Resistant starch was determined in the diets and glycemia monitored using glucose paper strips, the sample being blood obtained from the distal part of the rat’s tail. For the glycemic curve, glycemia was measured in the fasting state and during 90 minutes post-prandial. The results indicated there were no significant differences between the diets formulated with high, medium and low amylose, parboiled or conventionally prepared, with respect to fasting or post-prandial glycemia in Wistar rats.

Amylolytic activity in fruits: comparison of different substrates and methods using banana as model.

Several methodologies have been developed for cereal amylolytic activity estimation, but there is lack of information about the application of these methods for fruits. Mature green banana fruit can achieve 20% of starch content which is degraded during the ripening period in a complex process involving alpha- and beta-amylases and alpha-1,4 and alpha-1,6-glucosidases, besides phosphorylases that can compete for the same substrates. Methods used to determine total hydrolytic activity and individual activity of enzymes involved in starch breakdown were compared for banana extracts in several ripening stages. Total hydrolytic activity was measured by DNS and iodometric methods. Endoamylolytic activity on amylose-azure substrate was also evaluated. BPNPG7 and PNPG5 chromogenic substrates were used for alpha- and beta-amylase activities, respectively. The results showed that methods that depend on the use of thermal treatment or on inhibitors to inactivate one of the enzymes were not adequate. The use of p-nitrophenol derivatives seemed to be the most specific, reproducible, and easiest method employed for single alpha- and beta-amylases activities determination in complex tissues. The DNS and iodometric methods can be used only for initial screenings of total hydrolytic activity, because the nonspecific substrate used in these procedures allows the action of more than one enzyme simultaneously.