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Current Protocols in Plant Biology Sep 2018Maize is one the most widely cultivated crops worldwide and an important model system for the study of genetics and cytogenetics. Although the availability of a genome...
Maize is one the most widely cultivated crops worldwide and an important model system for the study of genetics and cytogenetics. Although the availability of a genome sequence has enabled new quantitative genomic studies, developing methods to isolate specific types of cells will enable useful approaches for transcriptomic analysis in the crop plant. Fluorescence-activated cell sorting (FACS) is a powerful technique for cell isolation and the study of transcriptional profiles from specific cell populations. The use of FACS on plant cells requires the generation of protoplasts by tissue digestion and cell wall removal. Although some protocols are available, they mainly focus on dicot species and obtaining sufficient protoplasts from inner tissue layers has been challenging in both monocots and dicots. Here, we report a new protocol that dramatically increases protoplast yield from maize for subsequent cell isolation by FACS. This protocol is efficient in generating protoplasts from root and shoot inner layers and can also be applied successfully to Arabidopsis thaliana. © 2018 by John Wiley & Sons, Inc.
Topics: Cell Wall; Flow Cytometry; Gene Expression Profiling; Plant Roots; Plant Shoots; Protoplasts; Zea mays
PubMed: 30138552
DOI: 10.1002/cppb.20072 -
Molecules (Basel, Switzerland) May 2018Take-all, caused by , is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, was transformed to express the...
Take-all, caused by , is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors-temperature, enzyme lysis time, and concentration of the lysing enzyme-were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 10⁷ protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46⁻54 transformants/μg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of . An optimised PEG-mediated protoplast transformation system for was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of and could be used to transform other fungi.
Topics: Gene Transfer Techniques; Phosphotransferases (Alcohol Group Acceptor); Polyethylene Glycols; Protoplasts; Saccharomycetales; Temperature; Transformation, Genetic; Triticum
PubMed: 29794975
DOI: 10.3390/molecules23061253 -
BMC Biotechnology Jul 2023L-lysine is widely used for feed and special diet products. The transformation of fermentation strains plays a decisive role in the development of these industries....
BACKGROUND
L-lysine is widely used for feed and special diet products. The transformation of fermentation strains plays a decisive role in the development of these industries. Based on the mutation breeding theory and metabolic engineering methods, this study aimed to improve the regeneration rate of high-lethality protoplasts by combining multiple mutagenesis and homologous cell fusion techniques to efficiently concentrate multiple dominant mutations and optimize the L-lysine production strain Escherichia coli QDW.
RESULTS
In order to obtain the best protoplasts, the optimal enzymolysis time was selected as 4 h. The optimal lysozyme concentration was estimated at 0.8 mg/mL, because the protoplast formation rate and regeneration rate reached 90% and 30%, respectively, and their product reached the maximum. In this study, it was necessary that UV mutagenesis be excessive to obtain an expanded mutation library. For high lethality protoplasts, under the premise of minimal influence on its recovery, the optimal time for UV mutagenesis of protoplasts was 7 min, and the optimal time for thermal inactivation of protoplasts at 85 ℃ was 30 min. After homologous fusion, four fusion strains of E. coli were obtained, and their stability was analyzed by flow cytometry. The L-lysine yield of QDW-UH3 increased by 7.2% compared with that of QDW in a fermentation experiment, which promoted the expression of key enzymes in L-lysine synthesis, indicating that the combination of ultraviolet mutagenic breeding and protoplast fusion technology improved the acid-production level of the fusion strain.
CONCLUSION
This method provides a novel approach for the targeted construction of microbial cell factories.
Topics: Fermentation; Protoplasts; Lysine; Escherichia coli; Regeneration
PubMed: 37452419
DOI: 10.1186/s12896-023-00792-8 -
Applied and Environmental Microbiology Oct 1986Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms...
Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.
Topics: Anaerobiosis; Animals; Benzaldehydes; Biodegradation, Environmental; Cell Wall; Fusobacterium; Microscopy, Electron; Protoplasts; Rumen; Streptococcus
PubMed: 3777921
DOI: 10.1128/aem.52.4.612-616.1986 -
International Journal of Molecular... Jun 2020Plants are sessile organisms that have a remarkable developmental plasticity, which ensures their optimal adaptation to environmental stresses. Plant cell totipotency is... (Review)
Review
Plants are sessile organisms that have a remarkable developmental plasticity, which ensures their optimal adaptation to environmental stresses. Plant cell totipotency is an extreme example of such plasticity, whereby somatic cells have the potential to form plants via direct shoot organogenesis or somatic embryogenesis in response to various exogenous and/or endogenous signals. Protoplasts provide one of the most suitable systems for investigating molecular mechanisms of totipotency, because they are effectively single cell populations. In this review, we consider the current state of knowledge of the mechanisms that induce cell proliferation from individual, differentiated somatic plant cells. We highlight initial explant metabolic status, ploidy level and isolation procedure as determinants of successful cell reprogramming. We also discuss the importance of auxin signalling and its interaction with stress-regulated pathways in governing cell cycle induction and further stages of plant cell totipotency.
Topics: Cell Differentiation; Cell Proliferation; Cellular Reprogramming; Mesophyll Cells; Plant Physiological Phenomena; Ploidies; Protoplasts; Signal Transduction; Totipotent Stem Cells
PubMed: 32545519
DOI: 10.3390/ijms21124195 -
Journal of Zhejiang University....This study aims to develop a method for isolating and purifying protoplasts/vacuoles from fresh leaves of the Cd hyperaccumulator plant species, Sedum alfredii. The...
This study aims to develop a method for isolating and purifying protoplasts/vacuoles from fresh leaves of the Cd hyperaccumulator plant species, Sedum alfredii. The results revealed that preheating cellulase and macerozyme at 50 °C for 5 min significantly accelerated the cell wall degradation. For the most optimal conditions for mesophyll protoplast isolation, the mixture of fresh leaves and cell lysates was followed by a 2-h-long vibration. The protoplast lysate for vacuole isolation was diluted, and 0.675 mmol/L was identified as the most appropriate 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS) level, in which S. alfredii large vacuoles are characterized by a high metal and malic acid content. For the best vacuole purification results, we established that 0.8 mol/L was the most optimal mannitol level in the vacuole buffer in terms of vacuole protection during centrifugation, whereas a Ficoll concentration of 0.10 g/ml was adopted in the density-gradient centrifugation.
Topics: Cadmium; Centrifugation; Centrifugation, Density Gradient; China; Germination; Malates; Mannitol; Plant Leaves; Plant Roots; Protoplasts; Sedum; Temperature; Vacuoles
PubMed: 28071001
DOI: 10.1631/jzus.B1600138 -
Applied and Environmental Microbiology Dec 1986Twenty-eight types of lysozyme-sensitive spores among seven Bacillus species representative of thermophiles, mesophiles, and psychrophiles were obtained spanning a... (Comparative Study)
Comparative Study
Twenty-eight types of lysozyme-sensitive spores among seven Bacillus species representative of thermophiles, mesophiles, and psychrophiles were obtained spanning a 3,000-fold range in moist-heat resistance. The resistance within species was altered by demineralization of the native spores to protonated spores and remineralization of the protonated spores to calcified spores and by thermal adaptation at maximum, optimum, and minimum sporulation temperatures. Protoplast wet densities, and thereby protoplast water contents, were obtained by buoyant density sedimentation in Nycodenz gradients (Nyegaard and Co., Oslo, Norway). Increases in mineralization and thermal adaptation caused reductions in protoplast water content between limits of ca. 57 and 28% (wet weight basis), and thereby correlated with increases in sporal heat resistance. Above and below these limits, however, increases in mineralization and thermal adaptation correlated with increases in sporal resistance independently of unchanged protoplast water contents. All three factors evidently contributed to and were necessary for heat resistance of the spores, but dehydration predominated.
Topics: Adaptation, Physiological; Bacillus; Bacillus cereus; Calcification, Physiologic; Desiccation; Geobacillus stearothermophilus; Hot Temperature; Protoplasts; Spores, Bacterial
PubMed: 3098170
DOI: 10.1128/aem.52.6.1242-1246.1986 -
Scientific Reports Feb 2021Citrus leprosis (CL) is a severe disease that affects citrus orchards mainly in Latin America. It is caused by Brevipalpus-transmitted viruses from genera Cilevirus and...
Citrus leprosis (CL) is a severe disease that affects citrus orchards mainly in Latin America. It is caused by Brevipalpus-transmitted viruses from genera Cilevirus and Dichorhavirus. Currently, no reports have explored the movement machinery for the cilevirus. Here, we have performed a detailed functional study of the p32 movement protein (MP) of two cileviruses. Citrus leprosis-associated viruses are not able to move systemically in neither their natural nor experimental host plants. However, here we show that cilevirus MPs are able to allow the cell-to-cell and long-distance transport of movement-defective alfalfa mosaic virus (AMV). Several features related with the viral transport were explored, including: (i) the ability of cilevirus MPs to facilitate virus movement on a nucleocapsid assembly independent-manner; (ii) the generation of tubular structures from transient expression in protoplast; (iii) the capability of the N- and C- terminus of MP to interact with the cognate capsid protein (p29) and; (iv) the role of the C-terminus of p32 in the cell-to-cell and long-distance transport, tubule formation and the MP-plasmodesmata co-localization. The MP was able to direct the p29 to the plasmodesmata, whereby the C-terminus of MP is independently responsible to recruit the p29 to the cell periphery. Furthermore, we report that MP possess the capacity to enter the nucleolus and to bind to a major nucleolar protein, the fibrillarin. Based on our findings, we provide a model for the role of the p32 in the intra- and intercellular viral spread.
Topics: Animals; Capsid Proteins; Citrus; Mites; Nucleocapsid; Plant Diseases; Plant Viral Movement Proteins; Plant Viruses; Protoplasts
PubMed: 33536554
DOI: 10.1038/s41598-021-82453-4 -
Viruses Apr 2019Plant-viroid interactions represent a valuable model for delineating structure-function relationships of noncoding RNAs. For various functional studies, it is desirable...
Plant-viroid interactions represent a valuable model for delineating structure-function relationships of noncoding RNAs. For various functional studies, it is desirable to minimize sample variations by using DNA, RNA, and proteins co-purified from the same samples. Currently, most of the co-purification protocols rely on TRI Reagent (Trizol as a common representative) and require protein precipitation and dissolving steps, which render difficulties in experimental handling and high-throughput analyses. Here, we established a simple and robust method to minimize the precipitation steps and yield ready-to-use RNA and protein in solutions. This method can be applied to samples in small quantities, such as protoplasts. Given the ease and the robustness of this new method, it will have broad applications in virology and other disciplines in molecular biology.
Topics: Arabidopsis; Arabidopsis Proteins; DNA, Plant; Genome, Plant; Host-Pathogen Interactions; Molecular Biology; Protoplasts; RNA, Plant; RNA, Viral; Viroids; Virus Replication
PubMed: 30987196
DOI: 10.3390/v11040324 -
Cells Jan 2021Plant cell walls (PCWs) form the outer barrier of cells that give the plant strength and directly interact with the environment and other cells in the plant. PCWs are...
Plant cell walls (PCWs) form the outer barrier of cells that give the plant strength and directly interact with the environment and other cells in the plant. PCWs are composed of several polysaccharides, of which cellulose forms the main fibrillar network. Enmeshed between these fibrils of cellulose are non-cellulosic polysaccharides (NCPs), pectins, and proteins. This study investigates the sequence, timing, patterning, and architecture of cell wall polysaccharide regeneration in suspension culture cells (SCC) of the grass species (). Confocal, superresolution, and electron microscopies were used in combination with cytochemical labeling to investigate polysaccharide deposition in SCC after protoplasting. Cellulose was the first polysaccharide observed, followed shortly thereafter by (1,3;1,4)-β-glucan, which is also known as mixed-linkage glucan (MLG), arabinoxylan (AX), and callose. Cellulose formed fibrils with AX and produced a filamentous-like network, whereas MLG formed punctate patches. Using colocalization analysis, cellulose and AX were shown to interact during early stages of wall generation, but this interaction reduced over time as the wall matured. AX and MLG interactions increased slightly over time, but cellulose and MLG were not seen to interact. Callose initially formed patches that were randomly positioned on the protoplast surface. There was no consistency in size or location over time. The architecture observed via superresolution microscopy showed similarities to the biophysical maps produced using atomic force microscopy and can give insight into the role of polysaccharides in PCWs.
Topics: Cell Wall; Cells, Cultured; Cellulose; Lolium; Protoplasts; Regeneration; Suspensions; Time Factors; Xylans; beta-Glucans
PubMed: 33440743
DOI: 10.3390/cells10010127