ABSTRACT
Size exclusion chromatography (SEC) and pyrene excimer formation (PEF) experiments were conducted to characterize the local density profile inside a glycogen sample before (Glycogen) and after (Gly-ß-LD) treatment with ß-amylase. These experiments were conducted to assess whether the density at the periphery of the glycogen particles was very high to limit access to proteins involved in the metabolism of glycogen as predicted by the Tier model or low as suggested by the Gilbert model. SEC analysis indicated that the density inside the Glycogen and Gly-ß-LD samples remained constant with particle size and was not affected by ß-amylolysis. Analysis of the PEF experiments conducted on the Glycogen and Gly-ß-LD samples labeled with 1-pyrenebutyric acid showed that the particles have a dense interior and loose corona. The conclusions reached by the SEC and PEF experiments agree with the Gilbert model and have implications for the association of glycogen ß-particles into larger α-particles.
Subject(s)
Chromatography, Gel , Glycogen , Particle Size , Pyrenes , Pyrenes/chemistry , Glycogen/chemistry , Chromatography, Gel/methods , beta-Amylase/metabolism , beta-Amylase/chemistry , FluorescenceABSTRACT
Sugars, synthesized by photosynthesis in source organs, are loaded and utilized as an energy source and carbon skeleton in sink organs, and also known to be important signal molecules regulating gene expression in higher plants. The expression of genes coding for sporamin and ß-amylase, the two most abundant proteins in storage roots of sweet potato, is coordinately induced by sugars. We previously reported on the identification of the carbohydrate metabolic signal-responsible element-1 (CMSRE-1) essential for the sugar-responsible expression of two genes. However, transcription factors that bind to this sequence have not been identified. In this study, we performed yeast one-hybrid screening using the sugar-responsible minimal promoter region of the ß-amylase gene as bait and a library composed only transcription factor cDNAs of Arabidopsis. Two clones, named Activator protein binding to CMSRE-1 (ACRE), encoding AP2/ERF transcription factors were isolated. ACRE showed transactivation activity of the sugar-responsible minimal promoter in a CMSRE-1-dependent manner in Arabidopsis protoplasts. Electric mobility shift assay (EMSA) using recombinant proteins and transient co-expression assay in Arabidopsis protoplasts revealed that ACRE could actually act to the CMSRE-1. Among the DEHYDRATION -RESPONSIVE ELEMENT BINDING FACTOR (DREB) subfamily, almost all homologs including ACRE, could act on the DRE, while only three ACREs could act to the CMSRE-1. Moreover, ACRE-homologs of Japanese morning glory also have the same property of DNA-binding preference and transactivation activity through the CMSRE-1. These findings suggested that ACRE plays an important role in the mechanism regulating the sugar-responsible gene expression through the CMSRE-1 conserved across plant species.
Subject(s)
Arabidopsis , Gene Expression Regulation, Plant , Ipomoea batatas , Plant Proteins , Promoter Regions, Genetic , Transcription Factors , beta-Amylase , Arabidopsis/genetics , Arabidopsis/metabolism , beta-Amylase/genetics , beta-Amylase/metabolism , Ipomoea batatas/genetics , Ipomoea batatas/metabolism , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolism , Promoter Regions, Genetic/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation/geneticsABSTRACT
KEY MESSAGE: The VaBAM3 cloned from Vitis amurensis can enhance the cold tolerance of overexpressed plants, but VaBAM3 knock out by CRISPR/Cas9 system weakened grape callus cold tolerance. In grape production, extreme cold conditions can seriously threaten plant survival and fruit quality. Regulation of starch content by ß-amylase (BAM, EC: 3.2.1.2) contributes to cold tolerance in plants. In this study, we cloned the VaBAM3 gene from an extremely cold-tolerant grape, Vitis amurensis, and overexpressed it in tomato and Arabidopsis plants, as well as in grape callus for functional characterization. After exposure to cold stress, leaf wilting in the VaBAM3-overexpressing tomato plants was slightly less pronounced than that in wild-type tomato plants, and these plants were characterized by a significant accumulation of autophagosomes. Additionally, the VaBAM3-overexpressing Arabidopsis plants had a higher freezing tolerance than the wild-type counterparts. Under cold stress conditions, the activities of total amylase, BAM, peroxidase, superoxide dismutase, and catalase in VaBAM3-overexpressing plants were significantly higher than those in the corresponding wild-type plants. Furthermore, sucrose, glucose, and fructose contents in these lines were similarly significantly higher, whereas starch contents were reduced in comparison to the levels in the wild-type plants. Furthermore, we detected high CBF and COR gene expression levels in cold-stressed VaBAM3-overexpressing plants. Compared with those in VaBAM3-overexpressing grape callus, the aforementioned indicators tended to change in the opposite direction in grape callus with silenced VaBAM3. Collectively, our findings indicate that heterologous overexpression of VaBAM3 enhanced cold tolerance of plants by promoting the accumulation of soluble sugars and scavenging of excessive reactive oxygen species. These findings provide a theoretical basis for the cultivation of cold-resistant grape and support creation of germplasm resources for this purpose.
Subject(s)
Gene Expression Regulation, Plant , Plant Proteins , Plants, Genetically Modified , Reactive Oxygen Species , Seedlings , Vitis , Vitis/genetics , Vitis/physiology , Vitis/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Reactive Oxygen Species/metabolism , Seedlings/genetics , Seedlings/physiology , Arabidopsis/genetics , Arabidopsis/physiology , Arabidopsis/metabolism , Cold Temperature , Solanum lycopersicum/genetics , Solanum lycopersicum/physiology , Solanum lycopersicum/metabolism , Sugars/metabolism , beta-Amylase/genetics , beta-Amylase/metabolism , Starch/metabolism , Cold-Shock Response/genetics , Cold-Shock Response/physiologyABSTRACT
Increasing the substrate concentration can effectively reduce energy consumption and result in more economic benefits in the industrial production of maltose, but this process remarkably increases the viscosity, which has a negative effect on saccharification. To improve saccharification efficiency, pullulanase is usually employed. In the conventional process of maltose production, pullulanase is added at the same time with ß-amylase or later, but this process seems inefficient when the substrate concentration is high. Herein, a novel method was introduced to enhance the maltose yield under high substrate concentration. The results indicated that the pullulanase pretreatment of highly concentrated maltodextrin solution for 2 h greatly affects the final conversion rate of ß-amylase-catalyzed saccharification. The maltose yield reached 80.95 %, which is 11.8 % above the control value. Further examination confirmed that pullulanase pretreatment decreased the number of branch points of maltodextrin and resulted in a high content of oligosaccharides. These linear chains were suitable for ß-amylase-catalyzed saccharification to produce maltose. This research offers a new effective and green strategy for starch sugar production.
Subject(s)
Polysaccharides , beta-Amylase , Maltose , Glycoside Hydrolases , Starch/chemistry , CatalysisABSTRACT
The ß-amylase (BAM) gene family encodes important enzymes that catalyze the conversion of starch to maltose in various biological processes of plants and play essential roles in regulating the growth and development of multiple plants. So far, BAMs have been extensively studied in Arabidopsis thaliana (A. thaliana). However, the characteristics of the BAM gene family in the crucial economic crop, cotton, have not been reported. In this study, 27 GhBAM genes in the genome of Gossypium hirsutum L (G. hirsutum) were identified by genome-wide identification, and they were divided into three groups according to sequence similarity and phylogenetic relationship. The gene structure, chromosome distribution, and collinearity of all GhBAM genes identified in the genome of G. hirsutum were analyzed. Further sequence alignment of the core domain of glucosyl hydrolase showed that all GhBAM family genes had the glycosyl hydrolase family 14 domain. We identified the BAM gene GhBAM7 and preliminarily investigated its function by transcriptional sequencing analysis, qRT-PCR, and subcellular localization. These results suggested that the GhBAM7 gene may influence fiber strength during fiber development. This systematic analysis provides new insight into the transcriptional characteristics of BAM genes in G. hirsutum. It may lay the foundation for further study of the function of these genes.
Subject(s)
Gossypium , beta-Amylase , beta-Amylase/genetics , Phylogeny , Multigene Family , Gene Expression Profiling , Plant Proteins/genetics , Plant Proteins/chemistryABSTRACT
In plant leaves, starch is composed of glucan polymers that accumulate in chloroplasts as the products of photosynthesis during the day; starch is mobilized at night to continuously provide sugars to sustain plant growth and development. Efficient starch degradation requires the involvement of several enzymes, including ß-amylase and glucan phosphatase. However, how these enzymes cooperate remains largely unclear. Here, we show that the glucan phosphatase LIKE SEX FOUR 1 (LSF1) interacts with plastid NAD-dependent malate dehydrogenase (MDH) to recruit ß-amylase (BAM1), thus reconstituting the BAM1-LSF1-MDH complex. The starch hydrolysis activity of BAM1 drastically increased in the presence of LSF1-MDH in vitro. We determined the structure of the BAM1-LSF1-MDH complex by a combination of cryo-electron microscopy, crosslinking mass spectrometry, and molecular docking. The starch-binding domain of the dual-specificity phosphatase and carbohydrate-binding module of LSF1 was docked in proximity to BAM1, thus facilitating BAM1 access to and hydrolysis of the polyglucans of starch, thus revealing the molecular mechanism by which the LSF1-MDH complex improves the starch degradation activity of BAM1. Moreover, LSF1 is phosphatase inactive, and the enzymatic activity of MDH was dispensable for starch degradation, suggesting nonenzymatic scaffold functions for LSF1-MDH in starch degradation. These findings provide important insights into the precise regulation of starch degradation.
Subject(s)
Arabidopsis Proteins , Arabidopsis , beta-Amylase , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Malate Dehydrogenase/metabolism , beta-Amylase/metabolism , Molecular Docking Simulation , Cryoelectron Microscopy , Starch/metabolism , Glucans/metabolism , Phosphoric Monoester Hydrolases/metabolism , Protein Serine-Threonine Kinases/metabolismABSTRACT
ß-amylase proteins (BAM) are important to many aspects of physiological process such as starch degradation. However, little information was available about the BAM genes in Annona atemoya, an important tropical fruit. Seven BAM genes containing the conservative domain of glycoside hydrolase family 14 (PF01373) were identified with Annona atemoya genome, and these BAM genes can be divided into four groups. Subcellular localization analysis revealed that AaBAM3 and AaBAM9 were located in the chloroplast, and AaBAM1.2 was located in the cell membrane and the chloroplast. The AaBAMs belonging to Subfamily I contribute to starch degradation have the higher expression than those belonging to Subfamily II. The analysis of the expression showed that AaBAM3 may function in the whole fruit ripening process, and AaBAM1.2 may be important to starch degradation in other organs. Temperature and ethylene affect the expression of major AaBAM genes in Subfamily I during fruit ripening. These expressions and subcellular localization results indicating ß-amylase play an important role in starch degradation.
Subject(s)
Annona , beta-Amylase , Annona/genetics , Annona/metabolism , Fruit/genetics , Fruit/metabolism , beta-Amylase/genetics , beta-Amylase/metabolism , Starch/genetics , Starch/metabolism , Gene Expression Regulation, Plant , Plant Proteins/genetics , Plant Proteins/metabolismABSTRACT
BACKGROUND: Hop creep continues to present an unresolved issue for the brewing industry, specifically stemming from those hops added to beer during fermentation. Hops have been found to contain four dextrin-degrading enzymes: alpha amylase, beta amylase, limit dextrinase, and an amyloglucosidase. One recent hypothesis predicts that these dextrin-degrading enzymes could originate from microbes rather than the hop plant itself. SCOPE AND APPROACH: This review begins by describing how hops are processed and used in the brewing industry. It will then discuss hop creep's origins with a new beer style, antimicrobial factors from hops and resistance mechanisms that bacteria use to counter them, and finally microbial communities that inhabit hops, focusing on whether they can produce the starch degrading enzymes which drive hop creep. After initial identification, microbes with possible links to hop creep were then run through several databases to search the genomes (if available) and for those specific enzymes. KEY FINDINGS AND CONCLUSIONS: Several bacteria and fungi contain alpha amylase as well as unspecified glycosyl hydrolases, but only one contains beta amylase. Finally, this paper closes with a short summary of how abundant these organisms typically are in other flowers.
Subject(s)
Humulus , beta-Amylase , Dextrins , alpha-Amylases , Beer/analysisABSTRACT
BACKGROUND: Grass leaf has been suspected of causing immunoglobulin (Ig)E-mediated immediate hypersensitivity reactions in humans and dogs. However, most studies in this area are case-control studies without in vitro data showing the involvement of IgE in the reaction. Laboratory studies have demonstrated the reactivity to a 50-55 kDa protein with clinical signs immediately after contact with grass leaf material. The clinical findings of dogs with atopic-like dermatitis immediately after contact with grass leaf material suggest the involvement of grass leaves as the allergen source. OBJECTIVES: This study was designed to test the IgE-reactivity of grass leaf proteins in dogs with clinical signs and positive scratch test results against grass leaf material. MATERIALS AND METHODS: The serum of 41 patients with a history of allergy and suspected to grass leaf material was immunoblotted against grass leaf extracts from five suspected grass species. The IgE-positive blots were separated with 2D gel electrophoresis and analysed with mass spectrometry (MS). Commercially supplied proteins were used to validate immunoblot activity. RESULTS: The serum of 25 dogs diagnosed with grass dermatitis had positive IgE-specific immunoblot against one or more grass leaf extracts. The MS data indicated a reactive band at 55 kDa to be beta-amylase or RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) large subunit (RbLS). All tested dog sera showed IgE-reactivity with beta-amylase and some with RbLS. CONCLUSIONS AND CLINICAL RELEVANCE: Canines with clinical signs of grass-related dermatitis had IgE-reactivity against grass leaf proteins. Serum IgE-reactivity to beta-amylase and RuBisCO large subunit may indicate that these proteins act as allergens, possibly causing pruritus and skin lesions.
Subject(s)
Dermatitis , Dog Diseases , Hypersensitivity, Immediate , Hypersensitivity , beta-Amylase , Dogs , Humans , Animals , Allergens/chemistry , Poaceae , Pollen , Immunoglobulin E , Ribulose-Bisphosphate Carboxylase , Hypersensitivity/diagnosis , Hypersensitivity/veterinary , Hypersensitivity, Immediate/veterinary , Plant Extracts , Dermatitis/veterinary , Dog Diseases/diagnosisABSTRACT
Starch is stored temporarily in the leaves during the day but degraded during the night. In this study, we investigated the relationship between diurnal changes in starch content in rice leaf blades and the mRNA levels of ß-amylase genes. In addition to the known plastid-type ß-amylases OsBAM2 and OsBAM3, OsBAM4, and OsBAM5 were also identified as plastid targeted proteins. In the leaf blades, starch contents, which reached its maximum at the end of day, showed two periods of marked decrease: from 18:00 to 21:00 and from 24:00 to 6:00. The expression of OsBAM2, OsBAM3, OsBAM4, and OsBAM5 was maintained at a low level from 18:00 to 21:00 but increased strongly after midnight. Furthermore, ß-amylase activity gradually increased after 21:00, reaching a maximum during the early morning. These results suggest that in rice leaf blades, ß-amylase plays an important role in starch degradation by being highly active from midnight to dawn.
Subject(s)
Oryza , beta-Amylase , Starch/metabolism , beta-Amylase/genetics , Oryza/genetics , Oryza/metabolism , Plant Leaves/genetics , Plant Leaves/metabolism , Carbohydrate MetabolismABSTRACT
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.
Subject(s)
Manihot , beta-Amylase , Drought Resistance , Manihot/metabolism , beta-Amylase/genetics , beta-Amylase/metabolism , alpha-Amylases/genetics , alpha-Amylases/metabolism , Droughts , Gene Expression Regulation, Plant , Stress, Physiological/genetics , Plant Proteins/genetics , Plant Proteins/metabolismABSTRACT
In this study, sweet potato ß-amylase (SPA) was modified by methoxy polyethylene glycol maleimide (molecular weight 5000, Mal-mPEG5000) to obtain the Mal-mPEG5000-SPA modified ß-amylase and the interaction mechanism between SPA and Mal-mPEG5000 was investigated. the changes in the functional groups of different amide bands and modifications in the secondary structure of enzyme protein were analyzed using infrared spectroscopy and circular dichroism spectroscopy. The addition of Mal-mPEG5000 transformed the random curl in the SPA secondary structure into a helix structure, forming a folded structure. The Mal-mPEG5000 improved the thermal stability of SPA and protected the structure of the protein from breaking by the surrounding. The thermodynamic analysis further implied that the intermolecular forces between SPA and Mal-mPEG5000 were hydrophobic interactions and hydrogen bonds due to the positive values of ΔHθ and ΔSθ. Furthermore, the calorie titration data showed that the binding stoichiometry for the complexation of Mal-mPEG5000 to SPA was 1.26, and the binding constant was 1.256 × 107 mol/L. The binding reaction resulted from negative enthalpy, indicating that the interaction of SPA and Mal-mPEG5000 was induced by the van der Waals force and hydrogen bonding. The UV results showed the formation of non-luminescent material during the interaction, the Fluorescence results confirmed that the mechanism between SPA and Mal-mPEG5000 was static quenching. According to the fluorescence quenching measurement, the binding constant (KA) values were 4.65 × 104 L·mol-1 (298K), 5.56 × 104 L·mol-1 (308K), and 6.91 × 104 L·mol-1 (318K), respectively.
Subject(s)
Ipomoea batatas , beta-Amylase , Circular Dichroism , Thermodynamics , Polyethylene Glycols , Maleimides , Protein Binding , Spectrometry, Fluorescence/methods , Binding Sites , Molecular Docking SimulationABSTRACT
Cold stress is a key climatic factor that limits grape productivity and quality. Although ß-amylase (BAM) is known to play an important role as a mediator of starch degradation under conditions of cold stress, the mechanism by which BAM regulates cold tolerance in grape remains unclear. Here, we identified VaBAM1 from Vitis amurensis and characterized its interactive regulating mechanism under cold stress in Arabidopsis thaliana and grape. VaBAM1-overexpressing A. thaliana plants (OEs) exhibited high freezing tolerance. Soluble sugar content and amylase activity were increased in OEs and VaBAM1-overexpressing grape calli (VaBAM1-OEs) under cold stress; however, they were decreased in grape calli in which VaBAM1 was edited using CRISPR/Cas9. The results of yeast two-hybrid, bimolecular fluorescence complementation, and pull-down experiments showed that serine/arginine-rich splicing factor 1 (VaSR1) interacted with VaBAM1. Furthermore, the expression of VaSR1 was opposite that of VaBAM1 in phloem tissue of Vitis amurensis during winter dormancy. In VaSR1-overexpressing grape calli (VaSR1-OEs), BAM activity and the expression levels of C-repeat binding transcription factor and cold response genes were all significantly lower than that in untransformed calli subjected to cold stress. Moreover, VvBAM1 was downregulated in VaSR1-OEs under cold stress. Overall, we identified that VaSR1 interacts with VaBAM1, negatively regulating BAM activity and resulting in decreased plant cold tolerance.
Subject(s)
Arabidopsis , Vitis , beta-Amylase , beta-Amylase/genetics , beta-Amylase/metabolism , Plant Proteins/chemistry , Arabidopsis/genetics , Cold-Shock Response , Carbohydrates , Gene Expression Regulation, Plant , Cold Temperature , Vitis/genetics , Vitis/metabolism , Plants, Genetically Modified/geneticsABSTRACT
ß-Amylase (BAM)-mediated starch degradation is a main source of soluble sugars that help plants adapt to environmental stresses. Here, we demonstrate that dehydration-induced expression of PtrBAM3 in trifoliate orange (Poncirus trifoliata (L.) Raf.) functions positively in drought tolerance via modulation of starch catabolism. Two transcription factors, PtrABF4 (P. trifoliata abscisic acid-responsive element-binding factor 4) and PtrABR1 (P. trifoliata ABA repressor 1), were identified as upstream transcriptional activators of PtrBAM3 through yeast one-hybrid library screening and protein-DNA interaction assays. Both PtrABF4 and PtrABR1 played a positive role in plant drought tolerance by modulating soluble sugar accumulation derived from BAM3-mediated starch decomposition. In addition, PtrABF4 could directly regulate PtrABR1 expression by binding to its promoter, leading to a regulatory cascade to reinforce the activation of PtrBAM3. Moreover, PtrABF4 physically interacted with PtrABR1 to form a protein complex that further promoted the transcriptional regulation of PtrBAM3. Taken together, our finding reveals that a transcriptional cascade composed of ABF4 and ABR1 works synergistically to upregulate BAM3 expression and starch catabolism in response to drought condition. The results shed light on the understanding of the regulatory molecular mechanisms underlying BAM-mediated soluble sugar accumulation for rendering drought tolerance in plants.
Subject(s)
Transcription Factors , beta-Amylase , Transcription Factors/genetics , Transcription Factors/metabolism , Drought Resistance , Amylases/genetics , Plant Proteins/metabolism , Abscisic Acid/metabolism , Carbohydrates , Droughts , Sugars , beta-Amylase/genetics , Starch/metabolism , Gene Expression Regulation, Plant , Plants, Genetically Modified/metabolism , Stress, PhysiologicalABSTRACT
Carbon reserves in cotton roots can be remobilized to support reproductive growth, thus potentially affecting cotton yield. However, the regulation of carbon remobilization in cotton roots and its relationship with cotton yield are still poorly understood. Plant population density (PPD) and mepiquat chloride (MC) have been hypothesized to affect the dynamics of nonstructural carbohydrate content and the resulting carbon remobilization in cotton roots through the regulation of carbohydrate metabolism enzyme activities. A mid-maturation cotton line 4003-6 was field-grown in 2019 and 2020. Three different levels of PPD (D1: 2.25 plants m-2, D2: 4.5 plants m-2, and D3: 6.75 plants m-2) and two levels of MC dosage (M0: 0 g hm-2, M1: 82.5 g hm-2) were combined to create six populations differing in terms of the source-sink relationship. The changes in the hexose, sucrose, and starch contents and the key carbon metabolic enzyme activities in cotton roots were examined during peak squaring (PS) to late boll opening (LB). The combination of the PPD of 6.75 plants m-2 and MC application (M1D3) exhibited the greatest cotton yield and reproductive biomass-to-leaf area ratio from peak flowering (PF) onwards. M1D3 presented the greatest total nonstructural carbohydrate (TNC) remobilization amount of 2.96 and 3.80 g m-2, the highest efficiency of 39.11% and 48.39%, and the largest gross contribution to seed cotton yield of 0.66% and 0.79% in 2019 and 2020, respectively. The three parameters were positively correlated with the seed cotton yield except for the remobilization rate in 2019. Unlike the other treatments, the greater carbohydrate content per unit ground area in M1D3 prior to the PF stage was attributed to the higher sucrose phosphate synthase (SPS) and ADP-glucose pyrophosphorylase (AGPase) activities during the PS to first flowering (FF) stages. Conversely, the greater α-amylase and ß-amylase activities in M1D3 at the PF stage accounted for the lower starch content at the EB stage, and the smaller vacuolar invertase (VIN) and cell wall invertase (CWIN) activities at the EB stage could be responsible for the lower hexose concentration at that time. The TNC remobilization amount had a positive association with the AGPase and SPS activities at the FF stage and with ß-amylase activity at the PF stage in cotton tap roots in 2019 and 2020. This study provides a cotton yield-improving alternative through the promotion of carbon remobilization in roots using certain agronomic practices.
Subject(s)
Carbon , beta-Amylase , Carbon/metabolism , beta-Fructofuranosidase/metabolism , Population Density , beta-Amylase/metabolism , Plant Roots/metabolism , Starch/metabolism , Carbohydrates , Gossypium/metabolismABSTRACT
Optimized source-sink interactions are determinants of both rice yield and quality. However, most source genes have not been well studied in rice, a major grain crop. In this study, OsBMY4 and OsISA3, the key ß-amylase and debranching enzymes that control transient starch degradation in rice leaves, were co-overexpressed in rice in order to accelerate starch degradation efficiency and increase the sugar supply for sink organs. Systematic analyses of the transgenic rice indicated that co-overexpression of OsBMY4 and OsISA3 not only promoted rice yield and quality, but also improved seed germination and stress tolerance. Moreover, since the OsBMY4 gene has not been characterized, we generated osbmy4 mutants using CRIPSR/Cas9 gene editing, which helped to reveal the roles of ß-amylase in rice yield and quality. This study demonstrated that specific modulation of the expression of some key source genes improves the source-sink balance and leads to improvements in multiple key traits of rice seeds.
Subject(s)
Oryza , beta-Amylase , Oryza/genetics , Oryza/metabolism , beta-Amylase/genetics , Seeds/genetics , Seeds/metabolism , Edible Grain/metabolism , Starch/metabolism , Gene Expression Regulation, Plant , Plant Proteins/genetics , Plant Proteins/metabolismABSTRACT
The bean fruit pericarp accumulates a significant amount of starch, which starts to be degraded 20 days after anthesis (DAA) when seed growth becomes exponential. This period is also characterized by the progressive senescence of the fruit pericarp. However, the chloroplasts maintained their integrity, indicating that starch degradation is a compartmentalized process. The process coincided with a transient increase in maltose and sucrose levels, suggesting that ß-amylase is responsible for starch degradation. Starch degradation in the bean fruit pericarp is also characterized by a large increase in starch phosphorylation, as well as in the activities of cytosolic disproportionating enzyme 2 (DPE2, EC 2.4.1.25) and glucan phosphorylase (PHO2, EC 2.4.1.1). This suggests that the rate of starch degradation in the bean fruit pericarp 20 DAA is dependent on the transformation of starch to a better substrate for ß-amylase and the increase in the rate of cytosolic metabolism of maltose.
Subject(s)
Arabidopsis , beta-Amylase , Maltose/metabolism , Fruit/metabolism , beta-Amylase/metabolism , Arabidopsis/metabolism , Starch/metabolismABSTRACT
ß-amylase (BAM) plays an important role in plant development and response to abiotic stresses. In this study, 5 DoBAM members were identified in yam (Dioscorea opposita Thunb.). A novel ß-amylase gene BAM1, (named DoBAM1), was isolated from yam varieties Bikeqi and Dahechangyu. The open reading frame (ORF) of DoBAM1 is 2806 bp and encodes 543 amino acids. Subcellular localization analysis indicates that DoBAM1 localizes to the cell membrane and cytoplasm. In the yam variety Dahechangyu, the starch content, ß-amylase activity, and expression of DoBAM1 were characterized and found to all be higher than in Bikeqi. DoBAM1 overexpression in tobacco is shown to promote the accumulation of soluble sugar and chlorophyll content and to increase the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ß-amylase. Under cold treatment, we observed the induced upregulation of DoBAM1 and lower starch content and malondialdehyde (MDA) accumulation than in WT plants. In conclusion, these results demonstrate that DoBAM1 overexpression plays an advanced role in cold tolerance, at least in part by raising the levels of soluble sugars that are capable of acting as osmolytes or antioxidants.
Subject(s)
Dioscorea , beta-Amylase , Dioscorea/genetics , Nicotiana/metabolism , beta-Amylase/genetics , beta-Amylase/metabolism , Starch/genetics , Carbohydrates , SugarsABSTRACT
Ethylene (ETH), as a key plant hormone, plays critical roles in various processes of plant growth and development. ETH has been reported to induce adventitious rooting. Moreover, our previous studies have shown that exogenous ETH may induce plant adventitious root development in cucumber (Cucumis sativus L.). However, the key genes involved in this process are still unclear. To explore the key genes in ETH-induced adventitious root development, we employed a transcriptome technique and revealed 1415 differentially expressed genes (DEGs), with 687 DEGs up-regulated and 728 DEGs down-regulated. Using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we further identified critical pathways that were involved in ETH-induced adventitious root development, including carbon metabolism (starch and sucrose metabolism, glycolysis/gluconeogenesis, citrate cycle (TCA cycle), oxidative phosphorylation, fatty acid biosynthesis, and fatty acid degradation), secondary metabolism (phenylalanine metabolism and flavonoid biosynthesis) and plant hormone signal transduction. In carbon metabolism, ETH reduced the content of sucrose, glucose, starch, the activity of sucrose synthase (SS), sucrose-phosphate synthase (SPS) and hexokinase (HK), and the expressions of CsHK2, pyruvate kinase2 (CsPK2), and CsCYP86A1, whereas it enhanced the expressions of ß-amylase 1 (CsBAM1) and ß-amylase 3 (CsBAM3). In secondary metabolism, the transcript levels of phenylalanine ammonia-lyase (CsPAL) and flavonoid 3'-monooxygenase (CsF3'M) were negatively regulated, and that of primary-amine oxidase (CsPAO) was positively regulated by ETH. Additionally, the indole-3-acetic acid (IAA) content and the expressions of auxin and ETH signaling transduction-related genes (auxin transporter-like protein 5 (CsLAX5), CsGH3.17, CsSUAR50, and CsERS) were suppressed, whereas the abscisic acid (ABA) content and the expressions of ABA and BR signaling transduction-related genes (CsPYL1, CsPYL5, CsPYL8, BRI1-associated kinase 1 (CsBAK1), and CsXTH3) were promoted by ETH. Furthermore, the mRNA levels of these genes were confirmed by real-time PCR (RT-qPCR). These results indicate that genes related to carbon metabolism, secondary metabolite biosynthesis, and plant hormone signaling transduction are involved in ETH-induced adventitious root development. This work identified the key pathways and genes in ETH-induced adventitious rooting in cucumber, which may provide new insights into ETH-induced adventitious root development and will be useful for investigating the molecular roles of key genes in this process in further studies.
Subject(s)
Cucumis sativus , beta-Amylase , Cucumis sativus/metabolism , Plant Growth Regulators/metabolism , beta-Amylase/genetics , beta-Amylase/metabolism , Plant Roots/metabolism , Indoleacetic Acids/metabolism , Ethylenes/metabolism , Abscisic Acid/metabolism , Starch/metabolism , Sucrose/metabolism , Carbon/metabolism , Fatty Acids/metabolism , Gene Expression Regulation, PlantABSTRACT
Starch is crucial to gluten-free bread (GFB) quality. Amylolytic enzymes modified starch in situ during breadmaking, impacting the quality of both fresh and stored bread. Here, the breadmaking and storage properties of GFB with added endo-amylases, exo-amylases and debranching enzymes were compared. The results demonstrated that the addition of amylolytic enzymes, especially exo-amylases and debranching enzymes, facilitated interactions between the enzymatically modified starch segments and structural agents to improve the structure and texture of fresh GFB. Exo-type ß-amylase produced lower crumb firming rate than endo-type α-amylase, although they produced similar retrogradation enthalpies and relative crystallinities. Maltooligosaccharide-forming enzymes with a non-strict endo-/exo-mode of cleavage and debranching enzymes markedly reduced the crumb firming rate and retrogradation enthalpy. Yet, maltooligosaccharide-forming enzymes produced a similar relative crystallinity as α-amylase, indicating that enzymes with endo-/exo-modes depended less on the reduction of starch recrystallization to retard crumb firming compared with debranching enzymes.