Protein-Protein Interactions Visualized by Bimolecular Fluorescence Complementation in Arabidopsis thaliana Protoplasts from Leaf.

Bimolecular fluorescence complementation (BiFC) is a powerful tool for studying protein-protein interactions in living cells. By fusing interacting proteins to fluorescent protein fragments, BiFC allows visualization of spatial localization patterns of protein complexes. This method has been adapted to a variety of expression systems in different organisms and is widely used to study protein interactions in plant cells. The Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana (N. benthamiana) leaf cells is widely used, but in this chapter, a method for BiFC assay using Arabidopsis thaliana protoplasts is presented.

Enhancement of cellulolytic enzyme production from intrageneric protoplast fusion of Aspergillus species and evaluating the hydrolysate scavenging activity.

BACKGROUND: Lignocellulosic biomass provides a great starting point for the production of energy, chemicals, and fuels. The major component of lignocellulosic biomass is cellulose, the employment of highly effective enzymatic cocktails, which can be produced by a variety of microorganisms including species of the genus Aspergillus, is necessary for its utilization in a more productive manner. In this regard, molecular biology techniques should be utilized to promote the economics of enzyme production, whereas strategies like protoplast fusion could be employed to improve the efficacy of the hydrolytic process. RESULTS: The current study focuses on cellulase production in Aspergillus species using intrageneric protoplast fusion, statistical optimization of growth parameters, and determination of antioxidant activity of fermentation hydrolysate. Protoplast fusion was conducted between A. flavus X A. terreus (PFFT), A. nidulans X A. tamarii (PFNT) and A. oryzae X A. tubingensis (PFOT), and the resultant fusant PFNT revealed higher activity level compared with the other fusants. Thus, this study aimed to optimize lignocellulosic wastes-based medium for cellulase production by Aspergillus spp. fusant (PFNT) and studying the antioxidant effect of fermentation hydrolysate. The experimental strategy Plackett-Burman (PBD) was used to assess how culture conditions affected cellulase output, the best level of the three major variables namely, SCB, pH, and incubation temperature were then determined using Box-Behnken design (BBD). Consequently, by utilizing an optimized medium instead of a basal medium, cellulase activity increased from 3.11 U/ml to 7.689 U/ml CMCase. The following medium composition was thought to be ideal based on this optimization: sugarcane bagasse (SCB), 6.82 gm; wheat bran (WB), 4; Moisture, 80%; pH, 4; inoculum size, (3 × 106 spores/ml); and incubation Temp. 31.8 °C for 4 days and the fermentation hydrolysate has 28.13% scavenging activities. CONCLUSION: The results obtained in this study demonstrated the significant activity of the selected fusant and the higher sugar yield from cellulose hydrolysis over its parental strains, suggesting the possibility of enhancing cellulase activity by protoplast fusion using an experimental strategy and the fermentation hydrolysate showed antioxidant activity.

Isolation, Purification, and Application of Protoplasts and Transient Expression Systems in Plants.

Protoplasts, derived from plant cells, exhibit remarkable totipotency and hold significant value across a wide spectrum of biological and biotechnological applications. These versatile applications encompass protein subcellular localization and interaction analysis, gene expression regulation, functional characterization, gene editing techniques, and single-cell sequencing. Protoplasts' usability stems from their inherent accessibility and their ability to efficiently incorporate exogenous genes. In this review, we provide a comprehensive overview, including details on isolation procedures and influencing factors, purification and viability assessment methodologies, and the utilization of the protoplast transient expression system. The aim is to provide a comprehensive overview of current applications and offer valuable insights into protoplast isolation and the establishment of transient expression systems in a diverse range of plant species, thereby serving as a valuable resource for the plant science community.

Preparation of leaf protoplasts from Populus (Populus × xiaohei T. S. Hwang et Liang) and establishment of transient expression system.

Poplar, as a typical woody plant, is an ideal raw material for the production of lignocellulose biofuel. However, the longer life cycle is not conducive to the rapid identification of poplar genes. At present, protoplasts have been used for gene function identification and high-throughput analysis in many model plants. In this paper, a simplified and efficient protoplast isolation and transient expression system of Populus (Populus × xiaohei T. S. Hwang et Liang) is described. Firstly, we proposed an efficient enzyme hydrolysis method for isolating protoplasts from leaves of Populus × xiaohei. Secondly, we optimized the conditions of protoplast transformation mediated by PEG, and established an efficient transient expression system of protoplasts of Populus × xiaohei. Finally, the subcellular localization of three identified Dof transcription factors (PnDof19, PnDof20 and PnDof30) was also observed in the nucleus by using this scheme, which proved that the method was feasible. In general, this efficient method of protoplast isolation and transformation can be used for the study of protein subcellular localization and can be applied to other fields of molecular biology, such as protein interaction, gene activation and so on.

Protoplasting and Fluorescence-Activated Cell Sorting of the Shoot Apical Meristem Cell Types.

The shoot apical meristems (SAMs) are located at the tip of the shoot apex. The SAM harbors stem cells that divide continually to provide cells for developing above-ground organs. Several important developmental events occur in SAMs, such as stem cell maintenance, organ differentiation, and flowering commitment which are under genetic control. The SAM is a collection of specialized cells organized in specific spatial domains. Deciphering the gene regulatory networks, guided by the developmental and environmental signals, in these discrete cell types is essential to decoding the SAM function. Here, I provide updates to the previously published protocols for the protoplasting and subsequent purification through fluorescence-activated cell sorting (FACS) of SAM cell types (Reddy, Fluorescence activated cell sorting of shoot apical meristem cell types. In: Riechmann JL, Wellmer F (eds) Flower development. Methods in molecular biology, vol 1110. Humana, New York, pp 315-321, 2014), which has provided genome-wide gene expression patterns at a single cell-type resolution.

Using A Protoplast Transformation System to Enable Functional Studies in Mangifera indica L.

Mangoes (Mangifera indica L.) are an important kind of perennial fruit tree, but their biochemical testing method and transformation technology were insufficient and had not been rigorously explored. The protoplast technology is an excellent method for creating a rapid and effective tool for transient expression and transformation assays, particularly in plants that lack an Agrobacterium-mediated plant transformation system. This study optimized the conditions of the protoplast isolation and transformation system, which can provide a lot of help in the gene expression regulation study of mango. The most beneficial protoplast isolation conditions were 150 mg/mL of cellulase R-10 and 180 mg/mL of macerozyme R-10 in the digestion solution at pH 5.6 and 12 h of digestion time. The 0.16 M and 0.08 M mannitol in wash solution (WI) and suspension for counting (MMG), respectively, were optimal for the protoplast isolation yield. The isolated leaf protoplasts (~5.4 × 105 cells/10 mL) were transfected for 30 min mediated by 40% calcium-chloride-based polyethylene glycol (PEG)-4000-CaCl2, from which 84.38% of the protoplasts were transformed. About 0.08 M and 0.12 M of mannitol concentration in MMG and transfection solutions, respectively, were optimal for protoplast viability. Under the florescence signal, GFP was seen in the transformed protoplasts. This showed that the target gene was successfully induced into the protoplast and that it can be transcribed and translated. Experimental results in this paper show that our high-efficiency protoplast isolation and PEG-mediated transformation protocols can provide excellent new methods for creating a rapid and effective tool for the molecular mechanism study of mangoes.

Redox basis of photosynthesis inhibition at supra-optimal bicarbonate in mesophyll protoplasts of Arabidopsis thaliana.

We examined the patterns of photosynthetic O2 evolution at 1 mM (optimal) and 10 mM (supra-optimal) bicarbonate in mesophyll protoplasts of Arabidopsis thaliana. The photosynthetic rate of protoplasts reached the maximum at an optimal concentration of 1 mM bicarbonate and got suppressed at supra-optimal levels of bicarbonate. We examined the basis of such photosynthesis inhibition by mesophyll protoplasts at supra-optimal bicarbonate. The wild-type protoplasts exposed to supra-optimal bicarbonate showed up signs of oxidative stress. Besides the wild-type, two mutants were used: nadp-mdh (deficient in chloroplastic NADP-MDH) and vtc1 (deficient in mitochondrial ascorbate biosynthesis). The protoplasts of the nadp-mdh mutant exhibited a higher photosynthetic rate and greater sensitivity to supra-optimal bicarbonate than the wild-type. The ascorbate-deficient vtc1 mutant had a low photosynthetic rate and no significant inhibition at high bicarbonate. The nadp-mdh mutants had elevated activities, protein, and transcript levels of key antioxidant enzymes. On the other hand, the antioxidant enzyme systems in vtc1 mutants were not much affected at supra-optimal bicarbonate. We propose that the inhibition of photosynthesis at supra-optimal bicarbonate depends on the redox state of mesophyll protoplasts. The robust antioxidant enzyme systems in protoplasts of nadp-mdh mutant might be priming the plants to sustain high photosynthesis at supra-optimal bicarbonate.

Galactoglucomannan oligosaccharides mitigate cadmium toxicity in maize protoplasts by improving viability and cell wall regeneration.

To avoid human health endangerment via the food chain, the investigation of Cd's effects on plant growth and development, and the discovery of various compounds that would mitigate the toxic effects of Cd, are essential. Galactoglucomannan oligosaccharides (GGMOs) are biologically active compounds, which improve the growth and development of plants. Therefore, the impact of GGMOs on the mitigation of Cd toxicity on maize (Zea mays L.) protoplasts was the main objective of this research. Here, protoplast viability, de novo cell wall regeneration on protoplasts' surface and Cd-uptake by protoplasts were studied. To study the influence of different treatments over time, the protoplasts were sampled on various days during the 14-day-long cultivation. The medium containing 2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine, and GGMOs in a 10-9 M concentration with a pH of 3.8 was found to be optimal for protoplast cultivation. The toxic effect of Cd2+, which was evident already on the 2nd day of cultivation, resulted in decreased protoplast viability, the de novo cell wall regeneration, and in increased Cd-uptake. However, the application of GGMOs on Cd-stressed protoplasts increased cell wall regeneration. Fully or partly regenerated cell walls decreased the uptake of Cd2+ through the plasma membrane and improved protoplast viability. This is the first study that confirmed that biologically active oligosaccharides promote cell wall regeneration on the protoplast surface in both non-stress and Cd-stress conditions.

Development of a protoplast isolation system for functional gene expression and characterization using petals of Camellia Oleifera.

Protoplasts preparation and purification have been frequently used in plant genetics and breeding studies, whereas application of protoplasts in woody plants is still in its infancy. Although transient gene expression using purified protoplasts is well-documented and widely used in model plants and agriculture crops, no instance of either stable transformation or transient gene expression in the woody plant Camellia Oleifera has as of yet been reported. Here, we developed a protoplast preparation and purification method using C. oleifera petals by optimizing osmotic condition with D-mannitol and polysaccharide-degrading enzyme concentrations for petal cell wall digestion, to reach a high efficiency of protoplast productivity and viability. The achieved protoplasts yield was approximately 1.42 × 107 cells per gram of petal material and the viability of protoplasts was up to 89%. In addition, we explored influencing factors of protoplast transformation, including concentrations of PEG4000 and plasmid DNA. The transformation efficiency of 81% could be reached under the optimized condition. This protoplast isolation and transient expression system were deployed to further identify the functional regulation of C. oleifera related genes and the subcellular distribution of their encoded products. In summary, the protoplast isolation and transient expression system we established using oil-tea tree petals is an efficient, versatile and time-saving system, being suitable for gene function characterization and molecular mechanism analysis.

Improving the regeneration rate of deep lethal mutant protoplasts by fusion to promote efficient L-lysine fermentation.

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.

Engineering and characterization of carbohydrate-binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts.

Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nkon ) and desorption (koff ) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.

Plant protoplasts in the age of synthetic biology.

Protoplasts, which are plant cells with their cell walls removed, have been used for decades in plant research and have been instrumental in genetic transformation and the study of various aspects of plant physiology and genetics. With the advent of synthetic biology, these individualized plant cells are fundamental to accelerate the 'design-build-test-learn' cycle, which is relatively slow in plant research. Despite their potential, challenges remain in expanding the use of protoplasts in synthetic biology. The capacity of individual protoplasts to hybridize to form new varieties, and to regenerate from single cells, creating individuals with new features is underexplored. The main objective of this review is to discuss the use of protoplasts in plant synthetic biology and to highlight the challenges to exploiting protoplast technologies in this new 'age of synthetic biology'.

Plant cellulose synthase membrane protein isolation directly from Pichia pastoris protoplasts, liposome reconstitution, and its enzymatic characterization.

Cellulose is synthesized by a plant cell membrane-integrated processive glycosyltransferase (GT) called cellulose synthase (CesA). Since only a few of these plant CesAs have been purified and characterized to date, there are huge gaps in our mechanistic understanding of these enzymes. The biochemistry and structural biology studies of CesAs are currently hampered by challenges associated with their expression and extraction at high yields. To aid in understanding CesA reaction mechanisms and to provide a more efficient CesA extraction method, two putative plant CesAs - PpCesA5 from Physcomitrella patens and PttCesA8 from Populus tremula x tremuloides that are involved in primary and secondary cell wall formation in plants were expressed using Pichia pastoris as an expression host. We developed a protoplast-based membrane protein extraction approach to directly isolate these membrane-bound enzymes, as confirmed by immunoblotting and mass spectrometry-based analyses. Our method gives 3-4-fold higher purified protein yield than the standard cell homogenization protocol. Our method resulted in liposome reconstituted CesA5 and CesA8 enzymes with similar Michaelis-Menten kinetic constants, Km = 167 µM, 108 µM and Vmax = 7.88 × 10-5 µmol/min, 4.31 × 10-5 µmol/min, respectively, in concurrence with the previous studies for enzymes isolated using the standard protocol. Taken together, these results suggest that CesAs involved in primary and secondary cell wall formation can be expressed and purified using a simple and more efficient extraction method. This protocol could help isolate enzymes that unravel the mechanism of native and engineered cellulose synthase complexes involved in plant cell wall biosynthesis.

Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene.

Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea's adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype.

Fast Identification of In Vivo Protein Phosphorylation Events Using Transient Expression in Leaf Mesophyll Protoplasts and Phos-tagTM SDS-PAGE.

Phosphorylation/dephosphorylation is a key posttranslational mechanism for signal transduction and amplification. Several techniques exist for assessing protein phosphorylation status, but each has its own drawbacks. The fast, straightforward, and low-tech approach described here uses transient overexpression of peptide-tagged proteins in Arabidopsis leaf mesophyll protoplasts and immunoblotting with Phos-tag™ SDS-PAGE and commercial anti-tag antibodies. We illustrate this with two relevant examples related to the SnRK1 protein kinase, which mediates metabolic stress signaling: Arabidopsis thaliana SnRK1 activation by T-loop (auto-)phosphorylation and SnRK1 phosphorylation of the Arabidopsis RAV1 transcription factor, which is involved in seed germination and early seedling development.

Automated, High-Throughput Protoplast Transfection for Gene Editing and Transgene Expression Studies.

In an era of cost-efficient gene synthesis and high-throughput construct assembly, the onus of scientific experimentation is on the rate of in vivo testing for the identification of top performing candidates or designs. Assay platforms that are relevant to the species of interest and in the tissue of choice are highly desirable. A protoplast isolation and transfection method that is compatible with a large repertoire of species and tissues would be the platform of choice. A necessary aspect of this high-throughput screening approach is the need to handle many delicate protoplast samples at the same time, which is a bottleneck for manual operation. Such bottlenecks can be mitigated with the use of automated liquid handlers for the execution of protoplast transfection steps. The method described within this chapter utilizes a 96-well head for simultaneous, high-throughput initiation of transfection. While initially developed and optimized for use with etiolated maize leaf protoplasts, the automated protocol has also been demonstrated to be compatible with other established protoplast systems, such as soybean immature embryo derived protoplast, similarly described within. This chapter also includes instructions for a sample randomization design to reduce the impact of edge effects, which might be present when microplates are used for fluorescence readout following transfection. We also describe a streamlined, expedient, and cost-effective protocol for determining gene editing efficiencies using the T7E1 endonuclease cleavage assay with a publicly available image analysis tool.

Optimization of Isolation and Transformation of Protoplasts from Uncaria rhynchophylla and Its Application to Transient Gene Expression Analysis.

Protoplast-based engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. Uncaria rhynchophylla is a traditional Chinese medicinal plant with a variety of pharmaceutically important indole alkaloids. In this study, an optimized protocol for U. rhynchophylla protoplast isolation, purification, and transient gene expression was developed. The best protoplast separation protocol was found to be 0.8 M D-mannitol, 1.25% Cellulase R-10, and 0.6% Macerozyme R-10 enzymolysis for 5 h at 26 °C in the dark with constant oscillation at 40 rpm/min. The protoplast yield was as high as 1.5 × 107 protoplasts/g fresh weight, and the survival rate of protoplasts was greater than 90%. Furthermore, polyethylene glycol (PEG)-mediated transient transformation of U. rhynchophylla protoplasts was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. The U. rhynchophylla protoplast transfection rate was highest (71%) when protoplasts were transfected overnight at 24 °C with the 40 µg of plasmid DNA for 40 min in a solution containing 40% PEG. This highly efficient protoplast-based transient expression system was used for subcellular localization of transcription factor UrWRKY37. Finally, a dual-luciferase assay was used to detect a transcription factor promoter interaction by co-expressing UrWRKY37 with a UrTDC-promoter reporter plasmid. Taken together, our optimized protocols provide a foundation for future molecular studies of gene function and expression in U. rhynchophylla.

Direct leaf-peeling method for areca protoplasts: a simple and efficient system for protoplast isolation and transformation in areca palm (Areca catechu).

BACKGROUND: Areca palm (Areca catechu) is a woody perennial plant of both economical and medicinal importance grown in tropical and subtropical climates. Yet, the molecular biology study of areca palm is extremely impeded by its unavailability of a transformation method. An efficient protoplast isolation and transformation system could be highly desirable to overcome this barrier. RESULTS: Here, we described a simple and efficient method for protoplast isolation and transformation from the perennial plant areca palm. A high yield of protoplasts (2.5 × 107 protoplasts per gram of fresh leaf tissues) was obtained from the fresh light green leaflet from the newly-emerged leaf digested overnight in the enzyme solution [2% (w/v) cellulase R10, 0.5% (w/v) macerozyme R10, 0.7 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] by the direct leaf-peeling method. The isolated areca protoplasts maintain viability of 86.6% and have been successfully transformed with a green fluorescent protein (GFP)-tagged plasmid (pGreen0029-GFP, 6.0 kb) via the polyethylene glycol (PEG)-mediated transformation. Moreover, the mannitol concentration (optimal: 0.7 M) was determined as a key factor affecting areca protoplast isolation. We also demonstrated that the optimal density of areca protoplast for efficient transformation was at 1.0-1.5 × 106 cells/ml. With the optimization of transformation parameters, we have achieved a relatively high transformation efficiency of nearly 50%. CONCLUSION: We have established the first efficient protocol for the high-yield isolation and transformation of areca palm protoplasts. This method shall be applied in various biological studies of areca palm, such as gene function analysis, genome editing, protein trafficking and localization and protein-protein interaction. In addition, the protoplast system offers a great genetic transformation approach for the woody perennial plant-areca palm. Moreover, the established platform may be applied in protoplast isolation and transformation for other important species in the palm family, including oil palm and coconut.

Protocols for Studying Protein Stability in an Arabidopsis Protoplast Transient Expression System.

Protein stability influences many aspects of biology, and measuring their stability in vivo can provide important insights into biological systems.This chapter describes in detail two methods to assess the stability of a specific protein based on its transient expression in Arabidopsis protoplasts. First, a pulse-chase assay based on radioactive metabolic labeling of cellular proteins, followed by immunoprecipitation of the protein of interest. The decrease in radioactive signal is monitored over time and can be used to determine the protein's half-life.Alternatively, we also present a nonradioactive assay based on the use of reporter proteins, whose ratio can be quantified. This assay can be used to determine the relative stability of a protein of interest under specific conditions.