The Construction of an Environmentally Friendly Super-Secreting Strain of Bacillus subtilis through Systematic Modulation of Its Secretory Pathway Using the CRISPR-Cas9 System.

Achieving commercially significant yields of recombinant proteins in Bacillus subtilis requires the optimization of its protein production pathway, including transcription, translation, folding, and secretion. Therefore, in this study, our aim was to maximize the secretion of a reporter α-amylase by overcoming potential bottlenecks within the secretion process one by one, using a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system. The strength of single and tandem promoters was evaluated by measuring the relative α-amylase activity of AmyQ integrated into the B. subtilis chromosome. Once a suitable promoter was selected, the expression levels of amyQ were upregulated through the iterative integration of up to six gene copies, thus boosting the α-amylase activity 20.9-fold in comparison with the strain harboring a single amyQ gene copy. Next, α-amylase secretion was further improved to a 26.4-fold increase through the overexpression of the extracellular chaperone PrsA and the signal peptide peptidase SppA. When the final expression strain was cultivated in a 3 L fermentor for 90 h, the AmyQ production was enhanced 57.9-fold. The proposed strategy allows for the development of robust marker-free plasmid-less super-secreting B. subtilis strains with industrial relevance.

Molecular Characteristics and Functional Identification of a Key Alpha-Amylase-Encoding Gene AMY11 in Musa acuminata.

Alpha-amylase (AMY) plays a significant role in regulating the growth, development, and postharvest quality formation in plants. Nevertheless, little is known about the genome-wide features, expression patterns, subcellular localization, and functional regulation of AMY genes (MaAMYs) in the common starchy banana (Musa acuminata). Twelve MaAMY proteins from the banana genome database were clustered into two groups and contained a conserved catalytic domain. These MaAMYs formed collinear pairs with the AMYs of maize and rice. Three tandem gene pairs were found within the MaAMYs and are indicative of putative gene duplication events. Cis-acting elements of the MaAMY promoters were found to be involved in phytohormone, development, and stress responses. Furthermore, MaAMY02, 08, 09, and 11 were actively expressed during fruit development and ripening. Specifically, MaAMY11 showed the highest expression level at the middle and later stages of banana ripening. Subcellular localization showed that MaAMY02 and 11 were predominately found in the chloroplast, whereas MaAMY08 and 09 were primarily localized in the cytoplasm. Notably, transient attenuation of MaAMY11 expression resulted in an obvious increase in the starch content of banana fruit, while a significant decrease in starch content was confirmed through the transient overexpression of MaAMY11. Together, these results reveal new insights into the structure, evolution, and expression patterns of the MaAMY family, affirming the functional role of MaAMY11 in the starch degradation of banana fruit.

Boosting resistant starch in rice: Bacterial inulin as a metabolic and glucose uptake modulator.

Bacillus stercoris PSSR12 (B. stercoris PE), an isolate from rice field soils, was identified via 16s rRNA sequencing. The synthesis of the inulin and inulin producing enzyme (IPE) in B. stercoris PE was verified using SDS-PAGE and FTIR. This study aimed to assess the impact of B. stercoris PE treatment on in vitro inhibition of α-amylase and α-glucosidase from traditional and commercial rice varieties of South India. Additionally, the study investigated enzymatic inhibition and mRNA expression of starch synthesis genes (RAmy1a, GBSSIa, SBEIIa, and SBEIIb). Glucose transporter gene expression (GLUT1 and GLUT4) patterns were analyzed in 3T3-L1 adipocytes to evaluate glucose uptake in B. stercoris PE treated rice varieties. The application of B. stercoris PE enhanced grain quality by imparting starch ultra-structural rigidity, inhibiting starch metabolizing enzymes, and inducing molecular changes in starch synthesis genes. This approach holds promise for managing type II diabetes mellitus and potentially reducing insulin dependence.

Two newly established and mutually related subfamilies GH13_48 and GH13_49 of the α-amylase family GH13.

Currently, the main α-amylase family GH13 has been divided into 47 subfamilies in CAZy, with new subfamilies regularly emerging. The present in silico study was performed to highlight the groups, represented by the maltogenic amylase from Thermotoga neapolitana and the α-amylase from Haloarcula japonica, which are worth of creating their own new GH13 subfamilies. This enlarges functional annotation and thus allows more precise prediction of the function of putative proteins. Interestingly, those two share certain sequence features, e.g. the highly conserved cysteine in the second conserved sequence region (CSR-II) directly preceding the catalytic nucleophile, or the well-preserved GQ character of the end of CSR-VII. On the other hand, the two groups bear also specific and highly conserved positions that distinguish them not only from each other but also from representatives of remaining GH13 subfamilies established so far. For the T. neapolitana maltogenic amylase group, it is the stretch of residues at the end of CSR-V highly conserved as L-[DN]. The H. japonica α-amylase group can be characterized by a highly conserved [WY]-[GA] sequence at the end of CSR-II. Other specific sequence features include an almost fully conserved aspartic acid located directly preceding the general acid/base in CSR-III or well-preserved glutamic acid in CSR-IV. The assumption that these two groups represent two mutually related, but simultaneously independent GH13 subfamilies has been supported by phylogenetic analysis as well as by comparison of tertiary structures. The main α-amylase family GH13 has thus been expanded by two novel subfamilies GH13_48 and GH13_49. KEY POINTS: • In silico analysis of two groups of family GH13 members with characterized representatives • Identification of certain common, but also some specific sequence features in seven CSRs • Creation of two novel subfamilies-GH13_48 and GH13_49 within the CAZy database.

Genome-Wide Identification and Expression Profiling of the α-Amylase (AMY) Gene Family in Potato.

Starch degradation provides energy and signaling molecules for plant growth, development, defense, and stress response. α-amylase (AMY) is one of the most important enzymes in this process. Potato tubers are rich in starch, and the hydrolysis of starch into sugar negatively impacts the frying quality of potato. Despite its importance, the AMY gene family has not been fully explored in potatoes. Here, we performed a detailed analysis of the StAMY gene family to determine its role in potato. Twenty StAMY genes were identified across the potato genome and were divided into three subgroups. The promoters of StAMY genes contained an array of cis-acting elements involved in growth and development, phytohormone signaling, and stress and defense responses. StAMY8, StAMY9, StAMY12, and StAMY20 were specifically expressed in mature tubers. Different StAMY gene family members tended to be upregulated in response to ß-aminobutyric acid (BABA), Phytophthora infestans (P. infestans), benzothiadiazole (BTH), heat, salt, and drought stress. In addition, different StAMY gene family members tended to be responsive to abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellic acid (GA3), and 6-benzylaminopurine (BAP) treatment. These results suggest that StAMY gene family members may be involved in starch and sugar metabolism, defense, stress response, and phytohormone signaling. The results of this study may be applicable to other starchy crops and lay a foundation for further research on the functions and regulatory mechanisms of AMY genes.

Automated optimization of the solubility of a hyper-stable α-amylase.

Most successes in computational protein engineering to date have focused on enhancing one biophysical trait, while multi-trait optimization remains a challenge. Different biophysical properties are often conflicting, as mutations that improve one tend to worsen the others. In this study, we explored the potential of an automated computational design strategy, called CamSol Combination, to optimize solubility and stability of enzymes without affecting their activity. Specifically, we focus on Bacillus licheniformis α-amylase (BLA), a hyper-stable enzyme that finds diverse application in industry and biotechnology. We validate the computational predictions by producing 10 BLA variants, including the wild-type (WT) and three designed models harbouring between 6 and 8 mutations each. Our results show that all three models have substantially improved relative solubility over the WT, unaffected catalytic rate and retained hyper-stability, supporting the algorithm's capacity to optimize enzymes. High stability and solubility embody enzymes with superior resilience to chemical and physical stresses, enhance manufacturability and allow for high-concentration formulations characterized by extended shelf lives. This ability to readily optimize solubility and stability of enzymes will enable the rapid and reliable generation of highly robust and versatile reagents, poised to contribute to advancements in diverse scientific and industrial domains.

Structural basis for the recognition of α-1,6-branched α-glucan by GH13_47 α-amylase from Rhodothermus marinus.

Glycoside hydrolase (GH) family 13 is among the main families of enzymes acting on starch; recently, subfamily 47 of GH13 (GH13_47) has been established. The crystal structure and function of a GH13_47 enzyme from Bacteroides ovatus has only been reported to date. This enzyme has α-amylase activity, while the GH13_47 enzymes comprise approximately 800-900 amino acid residues which are almost double those of typical α-amylases. It is important to know how different the GH13_47 enzymes are from other α-amylases. Rhodothermus marinus JCM9785, a thermophilic bacterium, possesses a gene for the GH13_47 enzyme, which is designated here as RmGH13_47A. Its structure has been predicted to be composed of seven domains: N1, N2, N3, A, B, C, and D. We constructed a plasmid encoding Gly266-Glu886, which contains the N3, A, B, and C domains and expressed the protein in Escherichia coli. The enzyme hydrolyzed starch and pullulan by a neopullulanase-type action. Additionally, the enzyme acted on maltotetraose, and saccharides with α-1,6-glucosidic linkages were observed in the products. Following the replacement of the catalytic residue Asp563 with Ala, the crystal structure of the variant D563A in complex with the enzymatic products from maltotetraose was determined; as a result, electron density for an α-1,6-branched pentasaccharide was observed in the catalytic pocket, and Ile762 and Asp763 interacted with the branched chain of the pentasaccharide. These findings suggest that RmGH13_47A is an α-amylase that prefers α-1,6-branched parts of starch to produce oligosaccharides.

Site-Directed Mutagenesis: Improving the Acid Resistance and Thermostability of Bacillus velezensis α-Amylase and Its Preliminary Feed Application.

The current study aimed to improve the acid resistance and thermostability of Bacillus velezensis α-amylase through site-directed mutagenesis, with a specific focus on its applicability to the feed industry. Four mutation sites, P546E, H572D, A614E, and K622E, were designed in the C domain of α-amylase, and three mutants, Mut1 (E), Mut2 (ED), and Mut3 (EDEE), were produced. The results showed that the specific activity of Mut3 was 50 U/mg higher than the original α-amylase (Ori) after incubation at 40 °C for 4 h. Compared to Ori, the acid resistance of Mut3 showed a twofold increase in specific activity at pH 2.0. Moreover, the results of preliminary feed hydrolysis were compared between Ori and Mut3 by designing three factors, three levels of orthogonal experiment for enzymatic hydrolysis time, feed quantity, and amount of amylase. It was observed that the enzymatic hydrolysis time and feed quantity showed an extremely significant difference (p < 0.01) in Mut3 compared to Ori. However, the amount of enzyme showed significant (p < 0.05) improvement in the enzymatic hydrolysis in Mut3 as compared to Ori. The study identified Mut3 as a promising candidate for the application of α-amylase in the feed industry.

Structural and functional insights of starch processing α-amylase from hyperthermophilic archaeon Pyrococcusabyssi.

The genomic screening of hyper-thermophilic Pyrococcus abyssi showed uncharacterized novel α-amylase sequences. Homology modelling analysis revealed that the α-amylase from P. abyssi consists of an N-terminal GH57 catalytic domain, α-amylase central, and C-terminal domain. Current studies emphasize in-silico structural and functional analysis, recombinant expression, characterization, structural studies through CD spectroscopy, and ligand binding studies of the novel α-amylase from P. abyssi. The soluble expression of PaAFG was observed in the E. coli Rosetta™ (DE3) pLysS strain upon incubation overnight at 18 °C in an orbital shaker. The optimum temperature and pH of the PaAFG were observed at 90 °C in 50 mM phosphate buffer pH 6. The Km value for PaAFG against wheat starch was determined as 0.20 ± 0.053 mg while the corresponding Vmax value was 25.00 ± 0.67 µmol min-1 mg-1 in the presence of 2 mM CaCl2 and 12.5 % glycerol. The temperature ramping experiments through CD spectroscopy reveal no significant change in the secondary structures and positive and negative ellipticities of the CD spectra showing the proper folding and optimal temperature of PaAFG protein. The RMSD and RMSF of the PaAFG enzyme determined through molecular dynamic simulation show the significant protein's stability and mobility. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various industrial applications.

Simultaneous improvement of thermostability and maltotriose-forming ability of a fungal α-amylase for bread making by directed evolution.

For applications in food industries, a fungal α-amylase from Malbranchea cinnamomea was engineered by directed evolution. Through two rounds of screening, a mutant α-amylase (mMcAmyA) was obtained with higher optimal temperature (70 °C, 5 °C increase) and better hydrolysis properties (18.6 % maltotriose yield, 2.5-fold increase) compared to the wild-type α-amylase (McAmyA). Site-directed mutations revealed that Threonine (Thr) 226 Serine (Ser) substitution was the main reason for the property evolution of mMcAmyA. Through high cell density fermentation, the highest expression level of Thr226Ser was 3951 U/mL. Thr226Ser was further used for bread baking with a dosage of 1000 U/kg flour, resulting in a 17.8 % increase in specific volume and a 35.6 % decrease in hardness compared to the control. The results were a significant improvement on those of McAmyA. Moreover, the mutant showed better anti-staling properties compared to McAmyA, as indicated by the improved sensory evaluation after 4 days of storage at 4 and 25 °C. These findings provide insights into the structure-function relationship of fungal α-amylase and introduce a potential candidate for bread-making industry.

Production of polyhydroxyalkanoates by engineered Halomonas bluephagenesis using starch as a carbon source.

A novel α-amylase Amy03713 was screened and cloned from the starch utilization strain Vibrio alginolyticus LHF01. When heterologously expressed in Escherichia coli, Amy03713 exhibited the highest enzyme activity at 45 °C and pH 7, maintained >50 % of the enzyme activity in the range of 25-75 °C and pH 5-9, and sustained >80 % of the enzyme activity in 25 % (w/v) of NaCl solution, thus showing a wide range of adapted temperatures, pH, and salt concentrations. Halomonas bluephagenesis harboring amy03713 gene was able to directly utilize starch. With optimized amylase expression, H. bluephagenesis could produce poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB). When cultured for PHB production, recombinant H. bluephagenesis was able to grow up to a cell dry weight of 11.26 g/L, achieving a PHB titer of 6.32 g/L, which is the highest titer that has been reported for PHB production from starch in shake flasks. This study suggests that Amy03713 is an ideal amylase for PHA production using starch as the carbon source in H. bluephagenesis.

Raw starch degrading alkaline α-amylase from Geobacillus kaustophilus TSCCA02: Production, characterization, and its potential for application as a detergent additive.

Geobacillus kaustophilus TSCCA02, a newly isolated strain from cassava (Manihot esculenta L.) rhizosphere soil in Thailand, showed maximum raw starch degrading enzyme (RSDE) activity at 252.3 ± 9.32 U/mL with cassava starch and peptone at 5.0 and 3.0 g/L, respectively. 16 S ribosomal RNA (rRNA) sequencing and phylogenetic tree analyses indicated that the TSCCA02 strain was closely related to G. kaustophilus. The crude RSDE had optimal activity at 60°C and pH 9.0. This enzyme degraded various kinds of starch including potato starch, cassava starch, rice flour, corn starch, glutinous rice flour, and wheat flour to produce sugar syrup at 60°C, as confirmed by scanning electron microscopy (SEM), thin-layer chromatography (TLC), and Fourier-transform infrared spectroscopy (FTIR). The major end products of starch hydrolysis were maltose and maltotriose with a small amount of glucose, confirming this enzyme as an α-amylase. The enzyme improved the washing efficiency of cotton fabric with commercial detergent. Results indicated the potential of alkaline α-amylase produced from a new isolate of G. kaustophilus TSCCA02 for application as a detergent additive on an industrial scale.

Discovery and characterization of the α-amylases cDNAs from Enchytraeus albidus shed light on the evolution of "Enchytraeus-Eisenia type" Amy homologs in Annelida.

Although enchytraeids have gained popularity in scientific research, fundamental questions regarding their feeding ecology and biology remain largely unexplored. This study investigates α-amylases, major digestive enzymes responsible for hydrolyzing starch and similar polysaccharides into sugars, in Enchytraeus albidus. Genetic data related to α-amylases is currently lacking for the family Enchytraeidae but also for the entire Annelida. To detect and identify coding sequences of the expressed α-amylase genes in COI-monohaplotype culture (PL-A strain) of E. albidus, we used classical "gene fishing" and transcriptomic approaches. We also compared coding sequence variants of α-amylase retrieved from transcriptomic data related to freeze-tolerant strains. Our results reveal that E. albidus possesses two distinct α-amylase genes (Amy I and Amy II) that are homologs to earthworm Eisenia fetida Ef-Amy genes. Different strains of E. albidus possess distinctive alleles of α-amylases with unique SNP patterns specific to a particular strain. Unlike Amy II, Amy I seems to be a highly polymorphic and multicopy gene. The domain architecture of the putative Amy proteins was found the same as for classical animal α-amylases with ABC-domains. A characteristic feature of Amy II is the lack of GHGA motif in the flexible loop region, similarly to many insect amylases. We identified "Enchytraeus-Eisenia type" α-amylase homologs in other clitellates and polychaetes, indicating the ancestral origin of Amy I/II proteins in Annelida. This study provides the first insight into the endogenous non-proteolytic digestive enzyme genes in potworms, discusses the evolution of Amy α-amylases in Annelida, and explores phylogenetic implications.

Transcription factor OsMYB30 increases trehalose content to inhibit α-amylase and seed germination at low temperature.

Low-temperature germination (LTG) is an important agronomic trait for direct-seeding cultivation of rice (Oryza sativa). Both OsMYB30 and OsTPP1 regulate the cold stress response in rice, but the function of OsMYB30 and OsTPP1 in regulating LTG and the underlying molecular mechanism remains unknown. Employing transcriptomics and functional studies revealed a sugar signaling pathway that regulates seed germination in response to low temperature (LT). Expression of OsMYB30 and OsTPP1 was induced by LT during seed germination, and overexpressing either OsMYB30 or OsTPP1 delayed seed germination and increased sensitivity to LT during seed germination. Transcriptomics and qPCR revealed that expression of OsTPP1 was upregulated in OsMYB30-overexpressing lines but downregulated in OsMYB30-knockout lines. In vitro and in vivo experiments revealed that OsMYB30 bound to the promoter of OsTPP1 and regulated the abundance of OsTPP1 transcripts. Overaccumulation of trehalose (Tre) was found in both OsMYB30- and OsTPP1-overexpressing lines, resulting in inhibition of α-amylase 1a (OsAMY1a) gene during seed germination. Both LT and exogenous Tre treatments suppressed the expression of OsAMY1a, and the osamy1a mutant was not sensitive to exogenous Tre during seed germination. Overall, we concluded that OsMYB30 expression was induced by LT to activate the expression of OsTPP1 and increase Tre content, which thus inhibited α-amylase activity and seed germination. This study identified a phytohormone-independent pathway that integrates environmental cues with internal factors to control seed germination.

Evidence that hematophagous triatomine bugs may eat plants in the wild.

Blood feeding is a secondary adaptation in hematophagous bugs. Many proteins are secreted in the saliva that are devoted to coping with the host's defense and to process the blood meal. Digestive enzymes that are no longer required for a blood meal would be expected to be eventually lost. Yet, in many strictly hematophagous arthropods, α-amylase genes, which encode the enzymes that digest starch from plants, are still present and transcribed, including in the kissing bug Rhodnius prolixus (Hemiptera, Reduviidae) and its related species, which transmit the Chagas disease. We hypothesized that retaining α-amylase could be advantageous if the bugs occasionally consume plant tissues. We first checked that the α-amylase protein of Rhodnius robustus retains normal amylolytic activity. Then we surveyed hundreds of gut DNA extracts from the sylvatic R. robustus to detect traces of plants. We found plant DNA in 8% of the samples, mainly identified as Attalea palm trees, where R. robustus are usually found. We suggest that although of secondary importance in the blood-sucking bugs, α-amylase may be needed during occasional plant feeding and thus has been retained.

Exploration of computational approaches to predict the structural features and recent trends in α-amylase production for industrial applications.

Amylases are biologically active enzymes that can hydrolyze starch to produce dextrin, glucose, maltose, and oligosaccharides. The amylases contribute approximately 30% to the global industrial enzyme market. The globally produced amylases are widely used in textile, biofuel, starch processing, food, bioremediation of environmental pollutants, pulp, and paper, clinical, and fermentation industries. The purpose of this review article is to summarize recent trends and aspects of α-amylases, classification, microbial production sources, biosynthesis and production methods, and its broad-spectrum applications for industrial purposes, which will depict the latest trends in α-amylases production. In the present article, we have comprehensively compared the biodiversity of α-amylases in different model organisms ranging from archaea to eukaryotes using in silico structural analysis tools. The detailed comparative analysis: regarding their structure, function, cofactor, signal peptide, and catalytic domain along with their catalytic residues of α-amylases in 16 model organisms were discussed in this paper. The comparative studies on alpha (α) amylases' secondary and tertiary structures, multiple sequence alignment, transmembrane helices, physiochemical properties, and their phylogenetic analysis in model organisms were briefly studied. This review has documented the recent trends and future perspectives of industrially important novel thermophilic α-amylases. In conclusion, this review sheds light on the current understanding and prospects of α-amylase research, highlighting its importance as a versatile enzyme with numerous applications and emphasizing the need for further exploration and innovation in this field.

Large scale microfluidic CRISPR screening for increased amylase secretion in yeast.

Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae, we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion.

Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization.

BACKGROUND: Raw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value. RESULTS: In this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter Pveg-PylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production. CONCLUSIONS: This study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis.

Genome-wide characterization and expression analysis of α-amylase and ß-amylase genes underlying drought tolerance in cassava.

BACKGROUND: Starch hydrolysates are energy sources for plant growth and development, regulate osmotic pressure and transmit signals in response to both biological and abiotic stresses. The α-amylase (AMY) and the ß-amylase (BAM) are important enzymes that catalyze the hydrolysis of plant starch. Cassava (Manihot esculenta Crantz) is treated as one of the most drought-tolerant crops. However, the mechanisms of how AMY and BAM respond to drought in cassava are still unknown. RESULTS: Six MeAMY genes and ten MeBAM genes were identified and characterized in the cassava genome. Both MeAMY and MeBAM gene families contain four genes with alternative splicing. Tandem and fragment replications play important roles in the amplification of MeAMY and MeBAM genes. Both MeBAM5 and MeBAM10 have a BZR1/BES1 domain at the N-terminus, which may have transcription factor functions. The promoter regions of MeAMY and MeBAM genes contain a large number of cis-acting elements related to abiotic stress. MeAMY1, MeAMY2, MeAMY5, and MeBAM3 are proven as critical genes in response to drought stress according to their expression patterns under drought. The starch content, soluble sugar content, and amylase activity were significantly altered in cassava under different levels of drought stress. CONCLUSIONS: These results provide fundamental knowledge for not only further exploring the starch metabolism functions of cassava under drought stress but also offering new perspectives for understanding the mechanism of how cassava survives and develops under drought.

Multiple loci with cumulative effects on late maturity α-amylase (LMA) in wheat.

MAIN CONCLUSION: The cumulative action of combinations of alleles at several loci on the wheat genome is associated with different levels of resistance to late maturity α-amylase in bread wheat. Resistance to late maturity α-amylase (LMA) in bread wheat (Triticum aestivum L.) involves a complex interaction between the genotype and the environment. Unfortunately, the incidence and severity of LMA expression is difficult to predict and once the trait has been triggered an unacceptably low falling number, high grain α-amylase may be the inevitable consequence. Wheat varieties with different levels of resistance to LMA have been identified but whilst some genetic loci have been reported, the mechanisms involved in resistance and the interaction between resistance loci requires further research. This investigation was focused on mapping resistance loci in populations derived by inter-crossing resistant wheat varieties or crossing resistant lines with a very susceptible line and then mapping quantitative trait loci. In addition to the previously reported locus on chromosome 7B for which a candidate gene has been proposed, loci were mapped on chromosomes 1B, 2A, 2B, 3A, 3B, 4A, 6A and 7D. These loci have limited effects on their own but have a cumulative effect in combination with each other. Further research will be required to determine the nature of the causal genes at these loci, to develop diagnostic markers and determine how the genes fit into the pathway that leads to the induction of α-AMY1 transcription in the aleurone of developing wheat grains. Depending on the target environmental conditions, different combinations of alleles may be required to achieve a low risk of LMA expression.