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First published online January 23, 2009; 10.1104/pp.108.133975 Plant Physiology 149:1231-1239 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Positive Fluorescent Selection Permits Precise, Rapid, and In-Depth Overexpression Analysis in Plant Protoplasts1,[C],[OA]Center for Genomics and Systems Biology, Biology Department, New York University, New York, New York 10003
Transient genetic modification of plant protoplasts is a straightforward and rapid technique for the study of numerous aspects of plant biology. Recent studies in metazoan systems have utilized cell-based assays to interrogate signal transduction pathways using high-throughput methods. Plant biologists could benefit from new tools that expand the use of cell culture for large-scale analysis of gene function. We have developed a system that employs fluorescent positive selection in combination with flow cytometric analysis and fluorescence-activated cell sorting to isolate responses in the transformed protoplasts exclusively. The system overcomes the drawback that transfected protoplast suspensions are often a heterogeneous mix of cells that have and have not been successfully transformed. This Gateway-compatible system enables high-throughput screening of genetic circuitry using overexpression. The incorporation of a red fluorescent protein selection marker enables combined utilization with widely available green fluorescent protein (GFP) tools. For instance, such a dual labeling approach allows cytometric analysis of GFP reporter gene activation expressly in the transformed cells or fluorescence-activated cell sorting-mediated isolation and downstream examination of overexpression effects in a specific GFP-marked cell population. Here, as an example, novel uses of this system are applied to the study of auxin signaling, exploiting the red fluorescent protein/GFP dual labeling capability. In response to manipulation of the auxin response network through overexpression of dominant negative auxin signaling components, we quantify effects on auxin-responsive DR5::GFP reporter gene activation as well as profile genome-wide transcriptional changes specifically in cells expressing a root epidermal marker.
It has been demonstrated that flow cytometric analysis and fluorescence-activated cell sorting (FACS) of plant protoplasts is practicable; moreover, this technique has yielded valuable results in a number of different fields of research (Harkins and Galbraith, 1984  ; Galbraith et al., 1995; Sheen et al., 1995). For instance, FACS of protoplasts from Arabidopsis (Arabidopsis thaliana) plants expressing tissue-specific fluorescent protein markers has been used to examine both basal and environmentally stimulated transcriptional profiles in particular cell types (Birnbaum et al., 2003; Brady et al., 2007; Dinneny et al., 2008; Gifford et al., 2008), and flow cytometry has been employed to analyze reactive oxygen species production and programmed cell death in tobacco (Nicotiana tabacum) protoplasts (Lin et al., 2006). A broad selection of fluorescence tools is available to study a plethora of physiological parameters in plants, for example, cis-regulatory elements fused to fluorescent proteins (Haseloff and Siemering, 2006), genetically encoded molecular sensors (Looger et al., 2005), or dye-based sensors (Haugland, 2002), can be used in combination with cytometry to measure diverse biological processes.
Here, we document the development of a protoplast transfection system that employs cytometry and a transient transformation vector harboring a fluorescent positive selection marker (pBeaconRFP; Fig. 1
). The notable advantage of this system is that it allows for the exclusive analysis of the transformed cells and facilitates high-throughput dual-color analysis. The new vector for use in this system is designed in such a way that it not only expresses a gene of interest but also expresses monomeric red fluorescent protein (mRFP). Furthermore, it is compatible with the Gateway recombinase-mediated cloning system, permitting fast and easy cloning. Because of its red emission spectrum, the mRFP marker can easily be used in combination with the commonly utilized GFP. We present two examples of this system's use in the analysis of an important signal transduction cascade involved in many aspects of plant development, namely the auxin perception pathway (Fig. 2
; Guilfoyle and Hagen, 2007
Transient transformation of protoplasts is a widely utilized tool in plant research that is swift and unproblematic. The technique can be used, for example, to monitor the regulation of promoter elements, to analyze gene expression or enzymatic activity in response to a variety of stimuli, to examine the roles of transcription factors or signal transduction cascade components, or to study the subcellular localization of proteins (Sheen, 2001 ; Yoo et al., 2007). As opposed to stable transformation of plants (Arabidopsis being the most commonly used platform), which generally takes months and requires the use of a transfecting agent (usually Agrobacterium tumefaciens), transfection of protoplasts can be achieved in just 1 d and entails only raw DNA and either a chemical- or electroporation-based transfection method. Additionally, transient transformation analyses can overcome problems encountered with stable overexpression, such as pleiotropic developmental effects or nonviability, when a cell-based assay is appropriate. However, due to the fact that protoplast transformation efficiency is never 100%, the results can be convoluted by the nontransformed cells.
Transformation efficiencies are often low and variable (Cummins et al., 2007
As a first example, we used the pBeaconRFP transient transformation system for the rapid analysis of a regulatory circuit by means of reporter gene readout. We overexpress dominant negative mutant isoforms of the Aux/IAA transcription factors (IAAnmII; Fig. 2; Tiwari et al., 2001
The second example of the application of the pBeaconRFP positive selection marker system involves examination of the transcriptional effects of the expression of IAAnmIIs in a specifically marked cell population (Fig. 4A ). We make use of protoplasts derived from a cell type-specific GFP marker line (PWER::GFP, the WEREWOLF promoter fused to GFP), which expresses primarily in atrichoblasts (Lee and Schiefelbein, 1999 ), transfected with two different IAAnmII isoforms. Subsequent genome-wide transcriptional profiling of auxin-treated and mock-treated IAAnmII-expressing cells makes it possible to distinguish distinctive patterns of gene expression regulated by the different mutant Aux/IAA isoforms.
The use of high-throughput cell-based screening methods in the study of regulatory networks has become a conventional and effective approach in animal systems (Müller et al., 2005 ; Palmer et al., 2006). Cytometric and FACS-based analyses have also been much more widespread and prolific in animal or microbiology research than in plant research. The combination of a selectable protoplast transformation system along with the use of cytometry now allows us to take these powerful techniques to a new level in plant research.
Modification of Reporter Gene Activation by Transient Overexpression
In order to demonstrate the use of the pBeaconRFP system to study signal transduction, we took advantage of the auxin-sensitive DR5::GFP reporter gene. DR5 is a highly active synthetic auxin response element created by Ulmasov and coworkers (1997)
In the original experiments (Ulmasov et al., 1997
It has been demonstrated previously that stabilizing mutations in domain II of Aux/IAA proteins lead to a repression of auxin-responsive reporter gene activation (Fig. 2; Tiwari et al., 2001 Here, we have constructed an mRFP-positive marker containing a Gateway-compatible transient transformation vector, pBeaconRFP (Fig. 1), and have cloned the dominant negative Aux/IAA isoforms IAA7mII and IAA19mII, provided by the Guilfoyle laboratory, into this vector. pMON999_mRFP was utilized as a control vector, expressing only mRFP. These vectors were used to transfect protoplasts derived from the roots of 1-week-old DR5::GFP Arabidopsis seedlings (Fig. 3A). After an overnight incubation, giving the transformed protoplasts the opportunity to start expressing the IAAnmIIs and mRFP, protoplast suspensions were treated with 5 µM indole-3-acetic acid (IAA) or mock treated with solvent and monitored cytometrically. Figure 3B shows the acquired cytometric data in a dot-plot format. Mock-transfected protoplast suspensions (suspensions that encountered the polyethylene glycol (PEG) transfection procedure without the addition of plasmid; see "Materials and Methods") displayed only a minor population expressing GFP when treated with solvent alone; this population likely represents protoplasts derived from the natural DR5::GFP-expressing auxin maxima of the root (i.e. the root tip and lateral root primordia). These suspensions exhibited a sizeable induction of GFP expression when treated with auxin, as expected. In suspensions transfected with the control vector, the induction of GFP expression was clearly apparent in both the nontransformed and RFP-positive, transformed protoplasts. In stark contrast, the auxin-induced GFP expression in suspensions overexpressing either IAA7mII or IAA19mII was only evident in the nontransformed cells and not perceptible in the RFP-positive protoplasts. Quantification of the GFP signal in RFP-positive cells (Fig. 3, C and D) demonstrates that there was an approximately 8-fold increase of GFP signal in protoplasts transformed with the control vector, whereas the protoplasts transformed with the dominant negative Aux/IAA isoforms exhibited no obvious induction. Interestingly, the quantification also showed that the GFP signal in mock-treated IAA7mII- and IAA19mII-expressing protoplasts was already less intense than in the protoplasts transformed with the control vector, a 1.7- and 2.6-fold repression, respectively (Fig. 3D). An independent experiment is presented, showing a time course analysis of GFP induction (Fig. 3E), reiterating the previous result and allowing examination of the kinetics of reporter gene activation.
These results corroborate previous results (Tiwari et al., 2001
In order to demonstrate an entirely novel use for the system, we used pBeaconRFP in combination with a cell type-specific GFP marker to isolate dual-labeled cells by FACS and to analyze the effects of overexpression in a particular cell population. An expansive collection of cell type-specific fluorescent markers is available to the plant research community (Lee et al., 2006 We have used pBeaconRFP to transiently express IAAnmIIs in protoplasts derived from the roots of PWER::GFP Arabidopsis seedlings. Following overnight incubation and a 3-h treatment with IAA or solvent alone, dual-labeled protoplasts were separated using FACS and transcriptionally profiled by means of microarray analysis (Fig. 4A). Protoplast suspensions were transfected with the pMON999_mRFP control vector, pBeaconRFP_IAA7mII, or pBeaconRFP_IAA19mII. Figure 4B shows microscopic images of a PWER::GFP protoplast suspension transfected with the control vector, demonstrating that there are protoplasts present in all four expected categories: blank, PWER::GFP alone, pMON999_RFP alone, and dual labeled. Untransfected wild-type and PWER::GFP protoplast suspensions as well as a wild-type protoplast suspension transfected with the control vector were employed to conservatively set up sorting gates in such a way that exclusively the dual-labeled protoplasts would be sorted (Fig. 4C). The experiment was performed in triplicate; nine separate transfections, 18 treatments, sorts, and microarrays. In corroboration with known auxin responses and our own data, the expression of Arabidopsis GH3.5, as measured by microarray, resembles the DR5::GFP expression measured in the previous experiment, displaying a drastically reduced auxin-induced increase in expression level and a basal repression of expression in protoplasts transformed with the IAAnmIIs (Figs. 3, D and E, and 4D). Furthermore, genes displaying a response to auxin in the protoplasts transformed with the control vector generally exhibited a dampened response in the protoplasts expressing IAAnmIIs (Table I ). Analysis of the data as a whole showed that the protoplasts transformed with the IAAnmIIs were fundamentally already very different compared with the protoplasts transformed with the control vector. Interestingly, although they were more similar to each other than to the control, there was also a substantial number of statistically significant gene expression differences between protoplasts expressing IAA7mII and IAA19mII (Fig. 4D; Table I).
These results provide a proof of concept for the feasibility of transcriptional profiling after transient protoplast transformation. This is now possible due to the fact that the system eliminates any contaminating effects of nontransformed cells. Furthermore, the dual-color cell-sorting approach makes it possible to analyze the effect of overexpression in a specific population of cells. In this case, the system allowed us to compare the outcome of expression of two highly homologous signal transduction cascade components, and the results indicate that IAA7 and IAA19 have both overlapping and unique downstream consequences in protoplasts derived from the Arabidopsis root epidermis. These results can be pursued to investigate mechanisms that lead to the specificity of auxin signal transduction. This demonstrates how the pBeaconRFP system can be used as a tool for rapid and high-throughput as well as in-depth analysis of genetic circuitry.
The system described here, making use of the pBeaconRFP positive selection marker vector in combination with flow cytometry and FACS, has several advantages over traditional protoplast transient transformation techniques. (1) The vectors containing a fluorescent positive marker make it possible to examine effects exclusively in the transformed protoplasts, thereby avoiding confounding of the results by nontransformed cells. (2) Functional data can be obtained even with protoplasts from tissues or species with intrinsically low transformation efficiencies. (3) The use of RFP as a positive marker allows cytometric analysis of transient gene expression in combination with systems employing any distinguishable fluorophores as readout. (4) FACS-based collection of transformed protoplasts also enables accurate use of any other nonfluorometric downstream analyses. (5) The system is Gateway compatible, making it quick and easy to clone genes of interest and amenable to high-throughput approaches (De Sutter et al., 2005 ). Making use of the pBeaconRFP vector and FACS-based collection of cells permits analysis not only of effects on GFP-reporter gene activation or transcriptional profiles, as demonstrated here, but also of any other measurable parameters, such as enzymatic activities and metabolite levels. In combination with cell identity markers, this system now also makes it possible to quickly analyze overexpression effects in a cell type-specific manner. Additionally, measuring effects in a particular cell population, as opposed to a heterogeneous mix of protoplasts, allows for a more defined and specific analysis. Moreover, there is the potential of measuring multiple parameters at once; for instance, one could measure the effect of the manipulation of upstream signal transduction elements on both mitogen-activated protein kinase activation and its ultimate downstream transcriptional responses. Of course, this system does not have to be used exclusively with flow cytometry or FACS; for example, it could also be used to select transformed protoplasts for individual analyses such as patch clamping or subcellular protein localization studies. Alternatively, a use in combination with more basic fluorometric analyses could be envisioned, such as microscopic analyses or assays performed with plate readers. Lastly, the system is conceptually well suited for high-throughput screening purposes (e.g. looking for genes that activate or inhibit the activation of a favorite reporter gene or complementation screens in mutant backgrounds). In conclusion, the technique described here opens up a wide field of possibilities not previously feasible in plant research.
Further development and enhancement of this system is ongoing. A transient silencing vector containing a positive selection marker will allow for RNA interference manipulations. Enhancement with glucocorticoid receptor protein fusion or a transcriptionally inducible system will make it possible to time the activation or overexpression of one's gene of interest (Moore et al., 2006 The pBeaconRFP vector will be made available through the Flanders Institute of Biotechnology (http://www.psb.ugent.be/gateway/), where the backbone originated. The microarray data have been deposited in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) database under accession number GSE13783.
Plant Materials and Treatment Seeds from wild-type (ecotype Columbia [Col-0]), DR5::GFP (Col-0; obtained from the Arabidopsis Biological Resource Center; stock no. CS9361), and PWER::GFP (Col-0; obtained from Dr. John Schiefelbein, University of Michigan) Arabidopsis (Arabidopsis thaliana) plants were sterilized by 5-min incubation with 96% ethanol followed by 20-min incubation with 50% household bleach and rinsing with sterile water. Seeds were plated on square 10-cm x 10-cm plates (Fisher Scientific) with MS agar (2.2 g L–1 Murashige and Skoog salts [Sigma-Aldrich], 1% [w/v] Suc, 1% [w/v] agar, 0.5 g L–1 MES hydrate [Sigma-Aldrich], pH 5.7, with KOH) on top of a sterile nylon mesh (NITEX 03-100/47; Sefar Filtration) to facilitate harvesting of the roots. Seeds were plated in two rows of approximately 150 seeds. Plates were vernalized for 2 d at 4°C in the dark and placed vertically in an Advanced Intellus environmental controller (Percival) set to 35 µmol m–2 s–1 and 22°C with an 18-h-light/6-h-dark regime. Protoplast suspensions were treated with 5 µM IAA (Sigma-Aldrich) or mock treated with solvent alone. A 20 mM IAA stock was dissolved in ethanol and stored at –20°C.
pMON999_mRFP was obtained from Dr. Joop Vermeer (Universiteit van Amsterdam). pBeaconRFP was constructed by PCR amplification of the 35S::mRFP::TNOS cassette from pMON999_mRFP with primers mRFP_F2 (5'-GAATTGCATATGCGTTCAAGCTTCTGCAGG-3') and mRFP_R (5'-TTAATACATATGCCCGGGGATCGATCC-3'), both with an NdeI restriction site (in boldface), using Phusion polymerase (New England Biolabs). The PCR product was ligated into the NdeI site of p2GW7.0 (http://www.psb.ugent.be/gateway/). The orientation of the insert was checked by PCR. A pZP211 plasmid containing 35S::HA-IAA7mII and a pUC18 plasmid containing PIAA19::HA-IAA19mII were obtained from Dr. Thomas Guilfoyle (University of Missouri). IAA7mII and IAA19mII were PCR amplified with primers IAA7_AttB1 (5'-AAAAAGCAGGCTATGATCGGCCAACTTATGAAC-3'), IAA7AttB2 (5'-AGAAAGCTGGGTTCAAGATCTGTTCTTGCAG-3'), IAA19AttB1 (5'-AAAAAGCAGGCTATGGAGAAGGAAGGACTC-3'), and IAA19AttB2 (5'-AGAAAGCTGGGTTCACTCGTCTACTCCTCTAG-3') and subsequently reamplified with primers AttB1 (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3') and AttB2 (5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3') using Phusion polymerase. The PCR products were recombined into pDONR221 using BP clonase and subsequently shuttled into pBeaconRFP with LR clonase (Invitrogen).
Protoplast isolation and PEG-mediated transfection were performed basically as described by the Sheen laboratory (http://genetics.mgh.harvard.edu/sheenweb/). Roots of 1-week-old seedlings were harvested with a scalpel and placed into a gently shaking flask with 100 mL of protoplasting solution for 3 h. Protoplasting solution was prepared as follows: 1.25% (w/v) cellulase (Yakult), 0.3% (w/v) macerozyme (Yakult), 0.4 M mannitol, 20 mM MES, 20 mM KCl, pH 5.7, with Tris-HCl, pH 7.5; heat for 10 min at 55°C, cool to room temperature; 0.1% (w/v) bovine serum albumin, 10 mM CaCl2, and 5 mM β-mercaptoethanol. The protoplast solution was filtered through a 40-µm cell strainer (BD Falcon), divided over 15-mL conical tubes, and centrifuged for 10 min at 150g. Pellets were washed once with transfection solution (0.4 M mannitol, 15 mM MgCl2 hexahydrate, 4 mM MES, pH 5.7, with KOH), centrifuged again, and resuspended in transfection solution with a final concentration of 4 x 106 protoplasts mL–1 (generally, we obtain between 8 x 103 and 1 x 104 protoplasts from 20 plates). Conical tubes (15 mL) were prepared for each transfection with 50 µg of plasmid DNA (10–20 µL) and 250 µL of protoplasts in transfection solution. PEG solution (250 µL; 40% [w/v] PEG 4000, 0.4 M mannitol, and 0.1 M CaCl2) was added, and the suspension was mixed by flicking the tube repeatedly. Suspensions were incubated for 15 min, after which the protoplasts were washed with 15 mL of incubation solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM MES, pH 5.7, with KOH), centrifuged, and resuspended in 1 mL of incubation solution. Protoplast suspensions were incubated overnight on 24-well plates in the dark.
Protoplast suspensions were cytometrically analyzed and sorted with a FACSAria (BD Biosciences) fitted with a 100-µm nozzle and using phosphate-buffered saline as a sheath fluid. The sheath pressure was set at 20 psi, and the defection plate voltage was set at 5,000 V (default "low" setting). A 488-nm Coherent Sapphire Solid State laser was used for excitation, and emission was measured at 530/30 nm for GFP and 610/20 nm for RFP. The photomultiplier tube voltage was set at 60 V for forward scatter, 159 V for side scatter, 350 V for GFP, and 335 V for RFP. The threshold value for event detection was set at 8,835 on forward scattering. The drop drive frequency was set to approximately 30 kHz, and the amplitude was set to approximately 45 V; the drop delay value was approximately 27 (these settings will vary slightly with day-to-day operation of the FACSAria). Data were processed using the FACSDiva 5.0.2 software (BD Biosciences). Compensation constraints were set to adjust for spectral overlap between GFP and RFP (GFP, minus 0.50% RFP; RFP, minus 17.91% GFP). Gates for sorting dual-labeled protoplasts were set up using blank (wild type), RFP-only (pMON999_mRFP-transfected wild type), and GFP-only (PWER::GFP) protoplast suspensions in such a way that the sorted dual-labeled protoplasts in the "double" gate would not be contaminated by blank, RFP-only, or GFP-only protoplasts (Fig. 4C).
Microscopic images of protoplasts mounted in a Bright-Line Hemacytometer (Hausser Scientific) were obtained with differential interference contrast, GFP, and Texas Red filters on an Eclipse 90i microscope (Nikon) running on Metamorph software (Molecular Devices).
Protoplasts were sorted directly into RNA extraction buffer, and RNA was extracted using an RNeasy Micro Kit with RNase-free DNase set according to the manufacturer's instructions (Qiagen). RNA was quantified with a Bioanalyzer (Agilent Technologies) and amplified and labeled with the WT-Ovation Pico RNA Amplification System and FL-Ovation cDNA Biotin Module V2, respectively (NuGEN). The labeled cDNA was hybridized, washed, and stained on an ATH-121501 Arabidopsis full-genome microarray using a Hybridization Control Kit, a GeneChip Hybridization, Wash, and Stain Kit, a GeneChip Fluidics Station 450, and a GeneChip Scanner (Affymetrix). Data were normalized using the MAS 5.0 method with a scaling factor of 250. Statistical analysis was performed as follows. We first filtered genes that showed expression below noise levels by removing genes whose average expression signal (among three replicates) never exceeded a threshold of 75 in any experiment. The data were subjected to two-way ANOVA (treatment x transient genetic background), and all genes that showed a significant effect at P < 0.05 at any level, including the interaction level, were taken as showing some response to experimental conditions (n = 7,145). These genes are shown on the heat map, which was generated with a log2 transformation of the data followed by row normalization. The heat map was generated in Matlab 7.6.0 (Mathworks). To test for gene expression differences in individual comparisons between the different treatments, we used a procedure that accounts for multiple testing (significance analysis of microarrays, two-class unpaired test, Wilcoxon statistic; q < 10% false discovery rate). In order to assess the effects of IAAnmII expression on auxin responses, the transcripts that showed a significant difference between mock-treated control vector and IAA-treated control vector (basal auxin response; n = 809) were then tested for their fold change response in experiments in which protoplasts were transiently transformed with pBeaconRFP_IAAnmII and mock treated or treated with auxin. Increases and decreases in average expression were converted to an absolute fold change to measure the overall effect of the overexpression on the basal auxin response. IAA7 and IAA19 were removed from analysis in their respective overexpressor samples.
We thank Joop Vermeer for the pMON999_mRFP plasmid, Tom Guilfoyle for the 35S::HA-IAA7mII and PIAA19::HA-IAA19mII plasmids, and John Schiefelbein for the PWER::GFP line. Received December 12, 2008; accepted January 20, 2009; published January 23, 2009.
1 This work was supported by the National Science Foundation (grant no. DBI 0519984).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Kenneth D. Birnbaum (ken.birnbaum{at}nyu.edu).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.133975 * Corresponding author; e-mail ken.birnbaum{at}nyu.edu.
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