•Large-scale analysis of transcription factor–cis-acting element interactions in plants, or the dissection of complex transcriptional regulatory mechanisms, requires rapid, robust and reliable systems for the quantification of gene expression.
•Here, we describe a new system for transient expression analysis of transcription factors, which takes advantage of the fast and easy production and transfection of Physcomitrella patens protoplasts, coupled to flow cytometry quantification of a fluorescent protein (green fluorescent protein). Two small-sized and high-copy Gateway® vectors were specifically designed, although standard binary vectors can also be employed.
•As a proof of concept, the regulation of BANYULS (BAN), a key structural gene involved in proanthocyanidin biosynthesis in Arabidopsis thaliana seeds, was used. In P. patens, BAN expression is activated by a complex composed of three proteins (TT2/AtMYB123, TT8/bHLH042 and TTG1), and is inhibited by MYBL2, a transcriptional repressor, as in Arabidopsis. Using this approach, two new regulatory sequences that are necessary and sufficient for specific BAN expression in proanthocyanidin-accumulating cells were identified.
•This one hybrid-like plant system was successfully employed to quantitatively assess the transcriptional activity of four regulatory proteins, and to identify their target recognition sites on the BAN promoter.
Transcription factors (TFs) are regulatory proteins found in all prokaryotic and eukaryotic organisms, including plants. TFs have been shown to control numerous facets of plant growth and development through the coordinated regulation of gene expression (Dubos et al., 2010). These regulatory mechanisms involve specific interactions between TFs and DNA motifs. To characterize TFs, their target DNA sequences are identified and their transcriptional activity is determined (i.e. activator or repressor). To identify the cis-regulatory elements that are targeted by specific TFs, a large number of constructs containing numerous versions of a promoter, with, in some cases, various combinations of regulators, are tested, as TFs can act alone or in combination with other protein partners.
The yeast one-hybrid system is a simple and efficient method for determining the relationships between proteins and target DNA. However, only a limited number of TFs can be tested at the same time because of the lack of selective markers (generally three; see Baudry et al., 2004). Furthermore, as a heterologous system, some yeast post-translational protein modifications can differ from those occurring in plants. Transient plant systems have been used extensively to study plant TF–promoter interactions because these approaches are fast and independent of any integration events into the genome (no position effects). These methods can be grouped into two categories based on the transformation protocol: the first relies on protoplast transfection and the second on tissues/cell transformation using agrobacteria. Protoplast preparation is time consuming and labour intensive (Hartmann et al., 1998). Leaf (Nicotiana benthamiana) or cotyledon infiltration is an efficient method, but is not high throughput (Berger et al., 2007; Marion et al., 2008). However, plant cultured cells may be suitable for high-throughput experiments (Berger et al., 2007; Marion et al., 2008), but this involves maintaining the cell cultures for periods of time, which is not a trivial task (Fukuda et al., 1994).
The combination of reporter genes together with transient plant expression systems is a powerful system for the investigation of TF activities on cis-target elements. Promoter activity can be assessed indirectly by measuring the product of a biochemical reaction, as is the case with the uidA (β-glucuronidase, GUS) and luciferase reporter genes, or directly by measuring the light emitted by a fluorescent protein (e.g. green fluorescent protein, GFP). The use of GFP is thus advantageous in terms of cost (as no expensive substrates are needed) and experimental time (less manipulation). The use of GFP as reporter gene is now widespread in the plant life sciences and allows the quantitative measurement of gene expression under various conditions. The analysis of transgene expression in plants by flow cytometry using GFP as a reporter gene has been the subject of much discussion and shows good potential for quantitative analysis (Sheen et al., 1995; Chiu et al., 1996; Galbraith et al., 1999a,b; Hagenbeek & Rock, 2001). For example, flow cytometry measurements (Fluorescence Activated Cell Sorting; FACS) allow the global analysis of an entire population of cells expressing GFP.
The moss Physcomitrella patens has recently emerged as a powerful model system, allowing fundamental questions of plant biology to be addressed (Prigge & Bezanilla, 2010). Although some higher plant-specific developmental aspects cannot be directly studied in moss, cellular processes and signalling can be successfully dissected in this system, establishing this moss as the yeast equivalent for plant research (Quatrano et al., 2007).
In this study, we show that P. patens protoplasts are a rapid and convenient model to study the regulation of gene expression. Gene expression was qualitatively and quantitatively studied using a method that combines the advantage of GFP as a marker of promoter activity with flow cytometry as a fast and reliable method for fluorescence measurements in cells. The method described herein allows very rapid sample processing, as only 2–3 d are necessary from the production of protoplasts to the final results.
As a proof of concept for an extensive analysis of TF activities, the regulation of Arabidopsis thaliana BANYULS (BAN), a key structural gene encoding an anthocyanidin reductase enzyme involved in the biosynthesis of the proanthocyanidin (PA) class of flavonoids, was studied (Xie et al., 2003). We validated, in P. patens protoplasts, that BAN expression is activated by a complex of three regulators, namely TT2/AtMYB123, TT8/bHLH042 and TTG1 (Baudry et al., 2004). Moreover, using P. patens protoplasts, we showed that two six-nucleotide-long cis-regulatory elements (G-box: CACGTG; MYB-core: CTGTTG) are the target recognition sites from which TT2, TT8 and TTG1 regulate BAN expression, at least partially. These cis-regulatory elements present on the BAN promoter were confirmed in planta in A. thaliana seeds, using uidA reporter gene activity (GUS). Finally, we confirmed that the activity of the TT2, TT8 and TTG1 complex can be counteracted by the transcriptional repressor MYBL2 (Dubos et al., 2008) in a quantitative manner.
Materials and Methods
Standard methods for plasmid construction were used. All PCRs were carried out using high-fidelity Phusion DNA polymerase (New England Biolabs, Evry, France), following the manufacturer’s instructions, and products were sequenced after recombination/cloning into their destination vectors. The primers used in this study are listed in Supporting Information Table S2. The Arabidopsis genes cloned were as follows: TT2/MYB123, At5g35550; TT8/bHLH042, At4g09820; TTG1, At5g24520; MYBL2, At1g71030; BAN, At1g61720.
pBS TPp-A A DNA fragment containing the Gateway® (Life Technologies, Carlsbad, CA, USA) recombination cassette, comprising the attR1 site, chloramphenicol resistance gene, ccdB counter selectable marker and attR2 site, was obtained after XbaI digestion of the pMDC140 vector (Curtis & Grossniklaus, 2003), and ligated into the XbaI-digested pCOR104-CaMVter plasmid (Proust et al., 2011) between the rice actin promoter and the 35S cauliflower mosaic virus terminator. The positive clones were selected on medium containing 100 μg ml−1 ampicillin and 12.5 μg ml−1 chloramphenicol.
pBS TPp-B A DNA fragment containing the Gateway® recombination cassette, comprising the attR1 site, chloramphenicol resistance gene, ccdB counter selectable marker, attR2 and GFP, was purified from the HindIII- and SacI-digested pGWB4 vector (Nakagawa et al., 2007). The pCOR104-CaMVter vector was digested with SmaI, blunt ended with Klenow and digested again with HindIII. The insert was then ligated into the digested HindIII-Blunt pCOR104-CaMVter to generate the pBS TPp-B vector. Positive clones were selected on medium containing 100 μg ml−1 ampicillin and 12.5 μg ml−1 chloramphenicol.
proOsActin:TT2, proOsActin:TT8, proOsActin:TTG1, proOsActin:MYBL2 The TT2, TT8, TTG1 and MYBL2 cDNAs were PCR amplified from 4-d-old silique cDNA (Ws ecotype) and subsequently recombined into the pDONR207 vector (BP Gateway® reaction) according to the manufacturer’s instructions. The cDNA clones were then inserted into the pBS TPp-A vector by LR reaction (Gateway®).
proBAN236:GFP and proBAN76:35Smini:GFP proBAN236 and proBAN76:35Smini were amplified from Ws genomic DNA, recombined into the pDONR207 vector and finally inserted into the pBS TPp-B vector (LR reaction; Gateway®).
proBAN236:uidA and proBAN76:35Smini:uidA pDONR207 vectors containing proBAN236 and proBAN76:35Smini were recombined into the binary pBI-101 Gateway® plasmid by an LR recombination (Baudry et al., 2006).
Mutagenized versions of the proBAN236 and proBAN76:35Smini promoters were obtained using the QuikChange® Site-Directed Mutagenesis Kit (Stratagene-Agilent, Massy, France), according to the manufacturer’s instructions. The corresponding pHISi and pDONR207 vectors were used as DNA templates for the reactions.
Yeast one-hybrid assay
Experiments were conducted with wild-type and mutated proBAN236 promoters, as described in Baudry et al. (2004). Briefly, after NcoI digestion, plasmids were inserted into the YM4271 yeast strain at the URA3 locus, before co-transfection with TT2 (pACTIIst vector) and TT8 (pAS2ΔΔ vector), or with the corresponding empty vectors. Yeast colonies were then assayed for HIS3 expression on medium lacking histidine.
Histochemical detection of GUS activity
GUS staining of seeds expressing proBAN236:uidA and proBAN763Smini:uidA (and mutated versions) constructs was performed as described by Debeaujon et al. (2003) in the presence of 0.5 or 2 mM potassium ferricyanide/potassium ferrocyanide.
The Gransden wild-type strain of P. patens was used in this study. Freshly fragmented protonema were inoculated on solid PPNH4 medium supplemented with 2.7 mM NH4-tartrate (Ashton & Cove, 1977; Ashton et al., 1979), overlaid with sterile cellophane disks (Cellulose type 325P, A.A. Packaging Limited, Preston, Lancashire, UK) in 90-mm Petri dishes, sealed with 3M Micropore™. Protonemal cultures were grown for 7 d at 24°C with a light regime of 16 h light : 8 h darkness at 80 μmol m−2 s−1 (adapted from (Schaefer & Zrÿd, 1997).
Moss protoplast preparation
Protoplasts were isolated from 6–7-d-old protonema by incubation for 1–2 h in 2% Driselase (Sigma D9515) dissolved in 0.47 M mannitol. The suspension was filtered successively through 63- and 40-μm stainless steel sieves. Protoplasts were sedimented by low-speed centrifugation (60 g for 5 min at 20°C) and washed twice with 0.47 M mannitol. Protoplasts were then resuspended at (0.6–1.2) × 106 protoplasts ml−1 in MMM solution (0.47 M mannitol, 15 mM MgCl2 and 0.1% 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.6) for transformation (adapted from Schaefer & Zrÿd, 1997; Trouiller et al., 2006).
Moss protoplast transformation
Transient transformations were carried out in 15-ml sterile tubes with 4.5 μg of DNA from each of the appropriate constructs, 300 μl of protoplast suspension and 300 μl of 40% (w/v) polyethylene glycol 4000 (PEG4000) solution, by heat shock for 7 min at 45°C. After 10 min at room temperature, the protoplasts were progressively diluted in 6.5 ml of liquid PPNH4 medium with 0.48 M mannitol, and left in the dark. Although a signal was observed after 24 h of incubation, in this study, the protoplasts were incubated for 48 h in order to obtain a better signal.
Flow cytometry measurements
Before flow cytometry analysis, protoplast suspensions were passed through a 30-μm mesh. Flow cytometry of live protoplasts expressing GFP was performed on a PARTEC CyFlow Space instrument (Partec S.A.R.L., Sainte Geneviève des Bois, France), using FLOMAX (Partec S.A.R.L., Sainte Geneviève des Bois, France) acquisition and analysis software, and a 488-nm solid sapphire 20-mW laser for excitation. The instrument was calibrated with calibration beads (3 μm, PARTEC reference 05-4008). The sheath fluid used was PARTEC reference 04-4007. GFP fluorescence was detected with an FITC 527-nm/30-nm band-pass filter (FL1 channel). Red chlorophyll-based fluorescence from living protoplasts was detected with a 610-nm/30-nm band-pass filter in the FL2 channel. The side light scatter (SSC) detector high voltage was set to 161.5 V. The photomultiplier tube voltages were adjusted to 275 V for FL1 and 475 V for FL2 (logarithmic amplification mode, four decades range, speed 4). For each sample, 40 000 events were analyzed on SSC to check the size of the protoplasts and to exclude debris. The protoplasts were then gated according to FL1 and FL2, as living GFP-expressing protoplasts should always show a red chlorophyll-based fluorescence. The weighted GFP fluorescence per population of cells was calculated as the product of the average fluorescence intensity of the population of cells above the background threshold. This threshold was set arbitrarily, based on a zero DNA transformed control compared with a positive control, as the fluorescence detection on FL1 shows a marked hook (Fig. S1).
Gating was drawn in such a way that the gated output was contaminated by nontransformed protoplasts (blank). The gate was drawn along a line of maximum GFP intensities for positive samples, when compared with protoplasts that were only transfected with proBAN236:GFP or proBAN76:35Smini:GFP as negative controls. The gate was unchanged until all measurements in the same experiment were completed. No compensation was performed. GFP-positive cells were normally distributed.
Images were obtained on a Zeiss Axioplan II epifluorescence microscope equipped with an HBO burner and using a GFP bandpass (459–490 nm BP 515–565 nm), a long-pass filter (459–490 nm LP 520 nm) and a 4′,6-Diamidino-2-Phenylindole (DAPI) filter (365–395 nm LP 397 nm). Images were obtained with a PROGRESS C3 digital camera (Jenoptik AG, Jena, Germany) and dedicated software. GUS experiments were analyzed according to Debeaujon et al. (2003).
Physcomitrella patens protoplast production, transformation and analysis
Our aim was to set up a fast and reliable system to monitor the interactions between TFs and their putative DNA targets. The moss P. patens is an emerging model for developmental and cellular studies. The system described here combines the use of moss protoplasts isolated from protonemal tissues, providing a large supply of single haploid cells for PEG-mediated transformation (Schaefer et al., 1991), with the robust quantification of fluorescence provided by flow cytometry (Hagenbeek & Rock, 2001; Ducrest et al., 2002). A general overview of the protocol is presented in Fig. 1. The production of protoplasts from moss tissues was obtained in < 3 h, which is much faster than that from plant cells/tissues (Dangl et al., 1987).
The quantity of plasmid needed for each transformation experiment (10–75 μg depending on the system used; Dangl et al., 1987; Kalbin et al. 1999; Kircher et al. 1999) is the principal drawback of the use of protoplasts, especially when employing low-copy binary vectors. Thus, to overcome this problem, we designed two small pBluescript-based vectors that are highly expressed in Escherichia coli, allowing between 30 and 60 transformations when extracted from a 100-ml bacterial culture (up to 300 μg of plasmid). These vectors were named pBS TPp-A and pBS TPp-B for TF (regulatory proteins) expression and promoter (regulatory sequences) activity detection, respectively (Fig. 2a). A Gateway® cassette containing the attR1 and attR2 recombination sites was introduced into both vectors to facilitate and accelerate the insertion of the TF and promoter sequences. TF expression in the pBS TPp-A vector is driven by the rice actin gene promoter, which has been shown to be well adapted for ubiquitous expression in P. patens (Horstmann et al., 2004).
As a result of the combination of rapid P. patens protoplast production, Gateway® cloning, high plasmid yield and the power of flow cytometry detection, our developed method appears to be extremely powerful for studying the regulation of gene expression. Indeed, sets of TFs and sets of promoter/regulatory sequences can be studied together in a medium- to high-throughput manner.
BAN promoter activity is induced by the TT2–TT8–TTG1 (MBW) ternary protein complex in P. patens protoplasts
In order to validate this new expression system, the regulation of the A. thaliana BAN gene was used as a model. BAN encodes an anthocyanidin reductase and is the first specific enzyme committed to PA (or condensed tannin) biosynthesis (Xie et al., 2003). PAs are flavonoids that specifically accumulate in the inner integument and the chalazal strand of the seed coat, where they participate in protecting the embryo and contribute to seed coat-imposed dormancy and seed longevity (Lepiniec et al., 2006). BAN expression has been studied extensively and, as such, offers a valuable system for the assessment of the application range and efficiency of use of moss P. patens protoplasts for gene expression studies (Debeaujon et al., 2003; Baudry et al., 2004). BAN promoter activity is specific to PA-accumulating cells when driven by a 236-bp-long DNA fragment located before the BAN start codon (proBAN236 minimal promoter; Fig. 3a). This specific pattern was also found in gain-of-function experiments using a 76-bp-long promoter fragment (−148 to −62 prior to the start codon; Fig. 3a) fused to the 35S cauliflower mosaic virus minimal promoter (proBAN76:35Smini). In Arabidopsis, BAN expression is also directly and positively regulated by a ternary MYB–BHLH–WDR (MBW) protein complex involving TT2/AtMYB123 (TRANSPARENT TESTA 2), TT8/AtBHLH042 and TTG1 (TRANSPARENT TESTA GLABRA 1: WD-repeat protein).
To test the robustness of our expression system, we introduced this complex transiently into P. patens, and checked for BAN expression. Protoplasts transfected with proBAN236:GFP alone displayed a low level of background fluorescence (FL1) when compared with the control (Figs 2d, 3b). When transfected together with the three members of the MBW complex, a strong fluorescence signal was detected in protoplasts (Figs 2d, 3b, S1). Interestingly, a weak, but significant, fluorescent signal was detected when TT2 and TT8 were transfected with proBAN236:GFP. This result confirms the role of TTG1 in the stabilization of the MBW complex (Baudry et al., 2004). No increase was observed when one member (or combinations of two members) of the MBW complex was transfected into the protoplasts (Fig. 3b). Similar results were obtained when proBAN76:35Smini:GFP was used (Fig. 4b).
Thus, using this approach, we successfully confirmed that TT2 and TT8, together with TTG1, can activate BAN promoter activity. The moss (P. patens) transient transformation system, coupled with flow cytometry, is therefore ideal for the fast and reproducible detection and analysis of complex protein–promoter interactions.
Identification of the binding sites of the TT2–TT8–TTG1 (MBW) complex on the BAN promoter using P. patens protoplasts
Although we were able to confirm that the MBW complex controls BAN expression using the moss protoplast system, the actual cis-regulatory sequences by which the TT2–TT8–TTG1 ternary complex regulates BAN promoter activity remained to be determined. Next, we therefore took advantage of the versatility of our method, associated with both its robustness and close relationship with higher plant cells, to address this new question.
A simple model for the regulation of BAN promoter activity can be formulated based on the work presented in Debeaujon et al. (2003), where a short 76-bp fragment was shown to be sufficient to specifically drive GUS activity in PA-accumulating cells. In this study, BHLH/MYC/G-box (CACGTG) and MYB-core (CTGTTG) putative regulatory elements were proposed to be the TT8 and TT2 binding sites, respectively. To assess the relative importance, if any, of these two putative cis-regulatory elements, wild-type and mutagenized versions of proBAN236 (Fig. 3a, Table S1) were used in yeast (proBAN236:Hisi) and P. patens (proBAN236:GFP) experiments (Fig. 3c,d). In yeast one-hybrid experiments, mutations at the G-box and MYB-core sites clearly disrupted yeast growth, indicating that these two sequences are necessary for activity of the MBW complex in this system (Fig. 3c). In P. patens protoplasts, both mutations had a negative effect on proBAN236 activity (Fig. 3d), confirming the results obtained in yeast. Together, these data indicate that the two analyzed cis-regulatory elements are the TT2 and TT8 binding sites from which the MBW complex activates proBAN236 transcription.
In order to confirm the involvement of these two regulatory motifs in the regulation of BAN expression, wild-type and mutated versions of proBAN76:35Smini (Fig. 4a and Table S1) were analyzed in both P. patens protoplasts (proBAN76:35Smini:GFP) and in planta (proBAN76:35Smini:uidA). In moss, the two mutated versions of the promoter showed decreased activity compared with the wild-type promoter (Fig. 4c). This result is consistent with observations in Arabidopsis seeds, where no GUS activity was detected in PA-accumulating cells (Fig. 4d), confirming previous suggestions (Debeaujon et al., 2003b).
MYBL2 negatively regulates MBW complex activity in a dose-dependent manner in P. patens protoplasts
MYBL2 is a negative regulator of flavonoid biosynthesis which belongs to the R3-MYB TF family (Dubos et al., 2008; Matsui et al., 2008). Increased MYBL2 expression in PA-accumulating cells inhibits PA accumulation in seeds, and the presence of MYBL2 counteracts MBW complex activities in Arabidopsis cells (Dubos et al., 2008). We relied on the repressor property of MYBL2 to examine whether the P. patens protoplast system is suitable for testing the repression activity of some TFs.
We found that the GFP intensity measured in moss protoplasts transfected with proBAN76:35Smini:GFP and all three transcriptional activators (TT2, TT8 and TTG1) was higher than in the protoplasts also co-transfected with MYBL2 (Fig. 5b). Of note, the observed decrease in GFP intensity was not a result of the addition of a fifth plasmid, as the addition of an empty vector used as control did not alter the GFP signal. Interestingly, we found that the negative effect of MYBL2 on the MBW complex activity was strongly correlated with the quantity of MYBL2 used in the experiment (Fig. 5b). Overall, this experiment shows that the repressive role of MYBL2 is conserved in P. patens, and demonstrates that quantitative promoter activity analysis can be carried out using the moss protoplast system coupled with flow cytometry measurements.
Plant TFs are regulatory proteins involved in the control of numerous facets of plant growth and development through the coordinated regulation of gene expression, which implies specific interactions between TFs and cis-element–DNA motifs. In this article, we describe a new strategy developed for the assessment of the relationship existing between plant TFs and their target DNA. This strategy allows the different problems inherent to the commonly used methods to be circumvented. Our approach is based on the use of the moss P. patens. We took advantage of the close phylogenetic relationship between moss and higher plants to develop this new approach that does not require specific skills. This close relationship could indeed be the source of false positive results if moss factors bind to the target promoter; conversely, it could also increase the robustness of the system by providing the necessary partners for suitable expression or stabilization of a given complex. This is, of course, the case with all ‘in vivo’ systems. Physcomitrella patens protoplasts, which are extremely fast and efficient to prepare and transfect, can thus be used in high-throughput experiments, employing already existing plant TF plasmid collections, to study the regulation of gene expression, within 2–3 d, starting from the moss culture.
We tested this method with several TF/cis-elements (this paper and unpublished data), but the technique can easily be scaled up to families of TFs and/or large sets of promoter sequences, using the flexibility of the Gateway® recombination system. In this case, the use of an automated loader coupled to fluorescence-activated cell sorting (FACS) would certainly increase the number of samples manipulated. In our system, the regulatory proteins were expressed under the control of a rice actin promoter (pACT) that is strongly expressed in moss protoplasts and does not require a selectable marker. This expression vector theoretically allows a large number of regulatory proteins to be tested in a single experiment.
The promoter activity of the studied gene was qualitatively and quantitatively analyzed using a method that combines the advantage of GFP as a quantitative marker of promoter activity with flow cytometry as a fast and reliable method for fluorescence measurements in cells (Sheen et al., 1995; Chiu et al., 1996; Galbraith et al., 1999a,b; Hagenbeek & Rock, 2001). Moreover, flow cytometry measurement allows the global analysis of the entire population of cells expressing the reporter gene and, as such, does not require any normalization with another reporter gene.
We successfully applied this method for the extensive analysis of the BAN promoter, a key structural gene encoding an anthocyanidin reductase (Xie et al., 2003). We were able to confirm, in a quantitative manner, that the MBW complex formed by TT2 (AtMYB123) and TT8 (bHLH042), together with TTG1, promotes BAN activity (Baudry et al., 2004), and that this transcriptional activation can be counteracted by MYBL2 (Dubos et al., 2008) in a dose-dependent manner in P. patens as in planta. Moreover, we successfully used the P. patens protoplast system to characterize the sequences (G-box, CACGTG; MYB-core, CTGTTG) involved in the MBW protein complex interaction with the BAN promoter, and from which this protein complex can activate BAN transcription in vivo. The involvement of these cis-regulatory elements in the regulation of BAN expression was confirmed in Arabidopsis seeds using uidA reporter gene activity (GUS).
In conclusion, the method presented here, which combines two existing robust techniques, that is moss (P. patens) protoplasts and GFP analysis by flow cytometry, is simple, fast, reliable, versatile and can be used in a high-throughput manner to study the interaction between regulatory proteins and regulatory sequences (i.e. transcriptional activity, protein–DNA interaction). It represents a plant-based one-hybrid-like screening system, paving the way for extensive promoter sequence analysis or the study of structure–function relationships between transcription factors and DNA.
This work was supported by the ANR ‘white’ program PLANT TF CODE and ANR KBBE STREG. The authors thank the ‘Plateforme de cytology et imagerie végétale (PCIV)’ for the technical support on flow cytometry. W.X.’s work was generously supported by the China Scholarship Council (CSC).