Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays

Background

Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function.

Methodology/Principal Findings

We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events.

Conclusions/Significance

This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.     

Preparation of leaf mesophyll protoplasts for transient gene expression in Brachypodium distachyon

Transient gene expression systems using protoplasts have been widely used for rapid functional characterization of genes in many plant species. Brachypodium distachyon has recently been employed as a model plant for studies on biofuel grass species and grass crops because of its small genome size, short life-span, and availability of efficient transformation systems. Here, we report the an efficient protocol for the preparation of leaf mesophyll protoplasts from Brachypodium seedlings. We also modified the polyethylene glycol (PEG)-mediated transformation procedure to optimize experimental conditions, such as duration of enzyme digestion, PEG incubation time, and plasmid DNA concentration and size. The green fluorescence protein (GFP)- and ??-glucuronidase (GUS)-coding genes were used as reporters to evaluate the feasibility of this transient expression system. We found that the yield of viable protoplasts was highest after 3 h of enzymatic digestion. Viability of enzyme-digested protoplasts was moderately maintained up to 24 h in Mmg preincubation solution. In addition, the highest transient expression of reporter genes was obtained when protoplasts were transformed with 20 ??g of plasmid DNA and incubated for 16 h. Together with the recent completion of the Brachypodium genome sequence, the Brachypodium transient expression system using leaf mesophyll protoplasts can be widely used for cellular, molecular, and biochemical studies of genes involved in carbon metabolism and signaling pathways mediating intrinsic and environmental cues.     

PEG-mediated transient gene expression and silencing system in maize mesophyll protoplasts: a valuable tool for signal transduction study in maize

Polyethylene glycol (PEG)-mediated transient gene expression and silencing in protoplasts is widely applied in model plants such as Arabidopsis thaliana and rice. Here, we developed an efficient transient gene expression system based on the PEG-mediated method both in etiolated and green maize mesophyll protoplasts. The results showed that both yellow fluorescent protein encoding gene and glucuronidase encoding gene were efficiently expressed in maize protoplasts. More importantly, double-stranded RNAs (dsRNAs) can also be transfected into maize protoplasts by the PEG-mediated method to specifically silence exogenous and endogenous genes. Our results showed that dsRNA can be used to knockdown both exogenous and endogenous gene expression. Furthermore, bimolecular fluorescence complementation system for the detection of protein–protein interactions in maize protoplasts was developed. We also overexpressed and knockdowned the mitogen-activated protein kinase encoding gene ZmMPK5 to investigate the role of ZmMPK5 in abscisic acid (ABA)-induced antioxidant defense in maize protoplasts. This method here we reported will be valuable for signal transduction study in maize.     

Simple and reliable system for transient gene expression for the characteristic signal sequences and the estimation of the localization of target protein in plant cell

The efficiency of transient gene expression in plants credibly demonstrated characteristics of gene functions in numerous studies. Two key strategies of transient expression became favorites among researchers: protoplast transfection and agroinfiltration. Each of them, alongside the advantages, has its own constraints. In this work, an easy, rapid, and reliable system for characterization of the signal sequences and determinations of target protein localization in a plant cell is proposed and tested. This system—called the AgI–PrI—implies production of protoplasts from plant tissues after agroinfiltration. Reliability of the proposed system for transient gene expression has been proved using characterized signal sequences in Nicotiana benthamiana cells. The corresponding protocol is less expensive and depends to a lesser degree on the professional skills in the area of protoplast isolation and transfection; furthermore, it may be applicable to other plant species with either available efficient methods of agroinfiltration and protoplast isolation or with the potential for one of the protocols to be supplemented. Thus, the AgI–PrI technique makes it possible to combine the advantages of two widely used methods for the transient gene expression in plants—agroinfiltration and protoplast isolation and transfection—and concurrently avoids their critical points.     

Efficient protoplast isolation and transient gene expression system for <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrid cultivar ‘Ruili Beauty’

With the release of the Phalaenopsis equestris (Schauer) Rchb.f. genome database, more in-depth studies of Phalaenopsis spp. will be carried out in the future. Transient gene expression in protoplasts is a useful system for gene function analysis, which is especially true for Phalaenopsis, whose stable genetic transformation is difficult and extremely time-consuming. In this study, juvenile leaves from aseptic Phalaenopsis seedlings were used as the starting material for protoplast isolation. After protocol refinement, the highest yield of viable protoplasts [5.94 × 10⁶ protoplasts g^?1 fresh weight (FW)] was achieved with 1.0% (w/v) Cellulase Onozuka R-10, 0.7% (w/v) Macerozyme R-10, and 0.4 M D-mannitol, with an enzymolysis duration of 6 h. As indicated by transient expression of green fluorescent protein (GFP), a transformation efficiency of 41.7% was achieved with 20% (w/v) polyethylene glycol (PEG-4000), 20 μg plasmid DNA, 2 × 10⁵ mL^?1 protoplasts, and a transfection duration of 30 min. The protocol established here will be valuable for functional studies of Phalaenopsis genes.     

Efficient transient expression and transformation of PEG-mediated gene uptake into mesophyll protoplasts of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

PEG-mediated transformation was used for gene delivery and evaluation of various parameters affecting the transient expression of a gene for ß-glucuronidase (gus) in mesophyll protoplasts of Capsicum annuum. Transient expression was found to be dependent on PEG concentration and exposure time of plasmid DNA to protoplasts as well as the amount of plasmid DNA. Maximum GUS activity was obtained when protoplasts were applied to 40% concentration and molecular weight was 6,000 of PEG solution with 30 min of exposure time. Protoplasts of pepper were transformed with a vector, pCAMBIA::Ac, which contained a pCAMBIA1302 T-DNA vector carrying a maize transposable element, Ac (activator), a selection marker HPT (hygromycin phosphotransferase), and a GFP-coding region driven by the 35S promoter in the presence of PEG. Approximately 30% of the protoplasts expressed GFP. Visibly transformed colonies were obtained from protoplasts after 2 months of culture and GFP was expressed. Southern hybridization confirmed the presence of Ac in the pepper genome.     

Overexpression of MinE gene affects the plastid division in cassava

The MinE protein plays an important role in plastid division. In this study, the MinE gene was isolated from the cassava (Manihot esculenta Crantz) genome. We isolated high quality and quantity protoplasts and succeed in performing the transient expression of the GFP-fused Manihot esculenta MinE (MeMinE) protein in cassava mesophyll protoplasts. The transient expression of MeMinE-GFP in cassava protoplasts showed that the MeMinE protein was located in the chloroplast. Due to the abnormal division of chloroplasts, overexpression of MeMinE proteins in cassava mesophyll protoplasts could result in fewer and smaller chloroplasts. Overexpression of MeMinE proteins also showed abnormal cell division characteristics and minicell occurrence in Escherichia coli caused by aberrant septation events in the cell poles.     

Characterization and Expression Analysis of a Glutathione Reductase Gene from Antarctic Moss Pohlia nutans

Glutathione reductase (GR) is a flavoprotein oxidoreductase and plays an important role in response to oxidative stresses in plants. A cDNA-encoding cytosolic GR [GenBank accession number GACA01029426, designated as Pohlia nutans glutathione reductase gene (PnGR)] was successfully cloned from Antarctic moss P. nutans. The full-length PnGR cDNA has 1,654 bp nucleotides with an open reading frame of 1,494 bp, encoding 497 amino acid residues. The deduced amino acid sequence of PnGR had 87.0 % identity with GR in Physcomitrella patens subsp. patens. The phylogenetic analysis showed that PnGR is clustered together with known cytosolic GR in other plants. In addition, the subcellular localization analysis by observing the transient expression of PnGR–green fluorescent protein fusion protein in Arabidopsis thaliana mesophyll protoplasts also revealed PnGR targeting to cytosol in plant cells. The expression patterns of PnGR under different abiotic stresses were determined by real-time PCR. Compared to the normal condition, the maximal mRNA accumulation of PnGR increased 3.82-fold at 4 °C, 2.92-fold at 10 °C, 4.14-fold with 200 mM NaCl, and 3.17-fold with drought stress, respectively. Together, our results suggested that the inducible PnGR might play an important role in Antarctic moss P. nutans acclimatizing to polar environment.     

Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici

Although transient gene expression using reporters such as green fluorescent protein is a versatile tool for examining gene functions and intracellular protein trafficking, the establishment of a highly efficient gene manipulation method remains a challenge in many plant species. A reliable transformation protocol has not yet been established for the three single-cell C₄ species, despite their potential of serving as model systems for their extraordinary C₄ photosynthetic metabolism. We report the first protocol optimized for isolating a large-scale and homogenous population of protoplasts from chlorenchyma cells of the single-cell C₄ species Bienertia sinuspersici. Cytochemical staining confirmed the preservation of the unusual subcellular compartmentation of organelles in chlorenchyma cells after cell wall digestion. Approximately 84% of isolated protoplasts expressed the reporter fluorescent protein following our optimized polyethylene glycol-mediated transfection procedures. Fluorescent fusion protein tagged with various intracellular sorting signals demonstrated potential use of the transient gene expression system in subcellular protein localization and organelle dynamics studies. Further applications of the current protoplast isolation and transfection techniques in understanding the novel single-cell C₄ photosynthetic mechanism are discussed.     

Transient gene expression and influence of promoters on foreign gene expression in Arabidopsis thaliana

Summary The influence of a variety of parameters was investigated on polyethylene glycol (PEG)-mediated transient nptII and gus gene expression in mesophyll protoplasts of Arabidopsis thaliana ecotype, Estland, in order to develop a suitable transient gene expression system. The investigation revealed that a combination of 20% PEG, incubation time of 15 min, 20–30 μg plasmid concentration per ml along with 50 μg carrier DNA m/l, and inclusion of calcium and magnesium ions during transfection followed by a culture period of 24 h registered maximum NPTII activity. Of the various promoters used for driving expression of the gus gene, the ubiquitin promoter from A. thaliana was the most efficient followed by 35S promoter of the CaMV and the actin promoter of rice. For comparison, similar studies in protoplasts of rice, wheat, and Brassica also revealed the differences in strength of these promoters. Arabidopsis ubiquitin promoter was the most effective in Brassica, and the rice actin1 promoter was the most effective in rice and wheat.     

An Efficient Protocol for Ulmus minor Mill. Protoplast Isolation and Culture in Agarose Droplets

We describe here an efficient and reproducible protocol for isolation and culture of protoplasts from Ulmus minor. Different sources of donor tissues were tested for protoplast isolation: callus and juvenile leaves from in vitro and greenhouse plants. Several combinations and concentrations of hydrolytic enzymes were used. Comparative tests between Cellulase Onozuka R10 and Cellulase Onozuka RS were made and the last one proved to be more efficient. Both the pectinases used, Macerozyme Onozuka R10 and Pectinase (Sigma^®), were efficient in protoplast isolation and there was no need for a more active pectinase. In vitro leaves proved to be the best source for protoplast isolation and produced an average of 3.96 × 10⁷ protoplasts per gram of fresh weigh. Elm mesophyll protoplasts were cultured using the advantageous method of agarose droplets and a modification of the Kao and Michayluk culture medium, using two plating densities (1 × 10⁵ and 2 × 10⁵ protoplasts ml^?1). Protoplast division and evolution into colonies and microcalli was promoted in the agarose droplets plated at 2 × 10⁵ protoplasts ml^?1. Ten weeks after protoplast culture initiation a plating efficiency of 2.7% was attained and the bigger microcalli, with at least 0.5 mm diameter, were transferred to a solid medium previously used for the production of embryogenic callus.     

Variation in gene transcription and protein for key enzymes of carbohydrate metabolism in poplar xylem with respect to growth phase

We compared gene expression levels for enzymes of carbohydrate metabolism in the twig xylem of two Populus species with the seasonal levels of starch and soluble sugars (sucrose, glucose, and fructose) and relative levels of the enzymes. Plants of Populus deltoides Bartr. ex Marsh and P. balsamifera L., 3–4 years old, were grown outside in Lubbock, TX, USA in 43 L pots. The xylem in the middle portion of the twigs was sampled during the dormant period (November–February), at bud break (for P. balsamifera), and during the growth flush (April–July). The gene expression for ADP-glucose pyrophosphorylase (AGPase), sucrose synthase (SuSy), and sucrose-phosphate synthase (SPS) generally coincided with the levels of the carbohydrates in whose metabolism these enzymes are involved. Gene expression for AGPase and its protein levels were high when the xylem starch content was high (growing period). However, P. balsamifera maintained high AGPase levels in dormant and growing twigs, unlike P. deltoides whose dormant twigs had low AGPase and low gene expression. Compared to growing twigs, gene expression for SuSy and SPS and their protein levels were higher in dormant twigs when soluble sugar content was higher. No down-regulation of these genes appears to occur when pools of the associated carbohydrates are high. Contrary to our expectation, the gene expression for β-amylase was highest in growing twigs when starch content was high. High β-amylase gene expression in growing twigs may be involved in maintaining a sufficient level of soluble sugars for growth through possibly controlling the extent of starch accumulation.     

A Transient RNA Interference Assay System Using Arabidopsis Protoplasts

Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.     

Protoplasts: a useful research system for plant cell biology, especially dedifferentiation

As protoplasts have the characteristics of no cell walls, rapid population growth, and synchronicity, they are useful tools for research in many fields, especially cellular biology (Table 1). This article is an overview that focuses on the application of protoplasts to investigate the mechanisms of dedifferentiation, including changes in hormone signals, epigenetic changes, and organelle distribution during the dedifferentiation process. The article also emphasizes the wide range of uses for protoplasts in studying protein positions and signaling during different stresses. The examples provided help to show that protoplast systems, for example the mesophyll protoplast system of Arabidopsis, represent promising tools for studying developmental biology. Meanwhile, specific analysis of protoplast, which comes from different tissue, has specific advantages and limitations (Table 2), and it can provide recommendations to use this system.