Plant Cell Reports
Plant Cell Reports (1993) t2:241 244
© Springer-Verlag1993
Transient expression in Arabidopsis thaliana protoplasts derived from rapidly established cell suspension cultures Jed H. Doelling and Craig S. Pikaard Biology Department and Center for Plant Science and Biotechnology, Box 1137, Washington University, One Brookings Drive, St. Louis, MO 63130, USA Received August 14, 1992/Revised version received December 14, 1992 - Communicated by J. M. Widholm
Abstract. Arabidopsis thaliana has emerged as a model species for the analysis of genes controlling plant development. However, its small size has impaired biochemical analyses, and the absence of a transient expression system has hampered promoter analysis. Here, we report a method for rapidly establishing A. thaliana suspension cultures that yield protoplasts that can be readily transfected. We have optimized transient expression conditions using a modified polyethylene glycol / calcium nitrate transformation protocol and a Cauliflower Mosaic Virus 35S promoter-luciferase reporter gene construct. Our methods permit isolation of large quantities of rapidly growing cells and analysis of Arabidopsis promoters in vivo in a homologous system. Abbreviations: CaMV, Cauliflower Mosaic Virus; 2,4D, 2,4-dichlorophenoxyacetic acid; MES, 2-(Nmorpholino)ethanesulfonic acid; PEG, polyethylene glycol.
Introduction
Arabidopsis thaliana is rapidly becoming the plant species of choice for molecular genetic studies of fundamental mechanisms controlling plant development. A large number of interesting mutations affecting such things as flower morphology, embryo development, and meristematic cell fate have been characterized (for review see Meinke, 1991). In many cases, the genes responsible for the mutant phenotypes have also been isolated and characterized. Studies of the expression of these genes, once isolated, has been done primarily in stably transformed Arabidopsis plants or in other easily transformed plants, such as tobacco. Production of transgenic plants is time consuming, and while necessary to evaluate tissue specificity and developmental regulation, is an inefficient means of analyzing cis-acting promoter elements recognized by the general transcriptional machinery. Transient expression of genes in plant protoplasts or in microprojectile bombarded intact cells is a much faster way Correspondence to: C. S. Pikaard
of analyzing these essential cis-acting sequences and has been widely used in established plant systems such as tobacco (Golds et. al, 1990). Our laboratory studies transcriptional regulation of the Arabidopsis ribosomal RNA genes, which are transcribed by RNA polymerase I. In animals, RNA polymerase I transcription is highly species-specific, such that the ribosomal genes of one species are generally not expressed in a transcription extract made from another species (Grummt et. al, 1982; reviewed by: Reeder, 1990; SollnerWebb and Mougey, 1991). Our preliminary results suggest that species-specificity of ribosomal gene transcription also occurs in plants (J.H. Doelling and C.S. Pikaard, unpublished). Consequently, we needed to develop a system for the transient expression of Arabidopsis genes in Arabidopsis cells. In this paper, we describe a tissue culture system for A. thaliana "Columbia" from which protoplasts are readily generated. These protoplasts support efficient transient expression of a luciferase reporter gene driven by the CaMV 35S promoter. We present data describing the optimization of several critical parameters for the transient expression system.
Materials and methods Establishment of cell cultures. Arabidopsis thaliana "Columbia" seeds (kindly provided by Dr. Elliot Meyerowitz) were sterilized by immersion in 95% ethanol in a 1.5 ml microfuge tube for 3-5 minutes, followed by immersion in 2.6% sodium hypochlorite solution for 30 minutes with occasional agitation. In a laminar flow hood, seeds were rinsed four times with sterile water and spread onto 0.8% agar plates containing 1X MS salts (Murashige and Skoog, 1962), 1X Gamborg vitamins (Gamborg et. al, 1968), 2 mg/1 2,4D, 0.05 mg/1 kinetin, and 2% glucose (all purchased from Sigma unless otherwise noted) or placed directly in liquid medium of the same composition excluding the agar. Plates were incubated in the light in a growth chamber with 16 hour, 24°C days and 8 hour, 21°C nights. Callus cultures were maintained by transferring the callus to fresh plates approximately once every three weeks. Liquid suspension cultures were initiated from about 300 mg of friable callus (fresh weight) inoculated into 24 ml of liquid medium in 125 ml Erlenmeyer flasks wrapped with aluminum foil to exclude light, Liquid cultures were grown on a gyrotory shaker at 85 rpm using the same temperature regime as the callus cultures. Optimal growth was obtained in a medium containing 3raM MES (USB), 2X Gamborg salts (Gamborg
242 et. al, 1968), 2X Gamborg vitamins, 1 mg/I 2,4D, 0.05 mg/1 Idnetin (hormones were filter-sterilized and added following autoclaving of the medium), and 2% sucrose (Fisher Biotech). The medium was adjusted to pH 5.7 with potassium hydroxide prior to autoclaving. Dense initial cultures were established from the dividing cell aggregates by replacing the old medium with fresh medium once a week. The old medium was most easily removed by pipet after first allowing the clumps of cells to settle. Small cell aggregates accumulated within a month and were used to seed multiple secondary cultures by allowing the largest cell clumps to settle, then transferring 10 ml of the cells still in suspension to a sterile, empty flask. After allowing these cells to settle, the old medium was removed and replaced with 24ml of fresh medium. The medium was replaced weekly for 3-4 weeks until the cultures grew to optimal density (one gram, fresh weight, per 24 ml of medium) for protoplast isolation. Protoplast isolation. Three days after the last medium exchange, approximately 2 grams (fresh weight) of cells in 48 ml of growth medium were poured into a deep petri dish (100 x 25 mm) and the medium removed. Fifteen ml. of filter-sterilized enzyme mix [1.1 g mannitol, 40 mg Maeerozyme R-10 (Serva), 150 mg cellulase "Onozuka R-10" (Serva), 70 mg Driselase, 7mM CaCI 2 (Fisher Biotech), and 3raM MES pH 5.7] was added to the cells. Digestion took place overnight in the dark at 21°C with gentle agitation (45 - 50 rpm) on a rotary shaker. Protoplasts were separated from undigested tissue by consecutive filtration through sterile 230 um, 104 um and 38 um stainless steel wire mesh (Small Parts, Inc. P.O. Box 4650 Miami Lakes, FL 33014-0650) into 50 ml conical tubes. Filtrates were centrifuged 10 minutes at room temperature, 26 x g, in a swinging bucket rotor. The protoplasts were washed twice in 0.34M mannitol, 0.07M CaC12, then resuspended in 10 ml 0.5M mannitol (not buffered) and counted in a hemacytometer. Typical protoplast yields were 2.5 x 107 protoplasts per gram fresh weight of suspension culture cells.
Optimal transfection conditions. Protoplasts stored in mannitol were pelleted and resuspended in ice cold MaMg solution (15mM MgCI 2 [FisherBioteeh], 5mM MES, 0.4M maunitol, pH 5.6) at a concentration of 1.5 x 107 protoplasts / ml and placed on ice. All further manipulations were performed on ice. To 100 ul MaMg solution in a 15 ml disposable conical tube, 100 ug of sheared calf thymus carrier DNA and 10 ug of pWB216 reporter gene construct were added and mixed well. One hundred microliters (1.5 x 106) of protoplast suspension was added to each tube using a wide bore pipet tip (sterile, disposable 0.2ml micropipetor tips with approximately 0.5 cm of the tip cut off) and mixed by pipetting. After a 10 minute incubation, 400 ul PEG CMS [40% PEG 8000 (Fisher Bioteeh), 0.4M mannitol, 0.1M Ca(NO3)2] was added to a final PEG concentration of 27%. Following a thirty minute incubation, this solution was diluted stepwise (diluted two-fold at each step) with 7 volumes ice cold W 5 solution (154mM NaCI [Fisherbiotec], 125mM CaCI2, 5mM KCI [USB], 5mM glucose, 1.5mM MES, 0.4M mannitol, pH 5.8). Protoplasts were pelleted at 26 x g, 4°C, and the pellets washed once with a solution consisting of 2 ml 0.5M mannitol and 0.5 ml W 5 solution. Protoplasts were resuspended in 3 ml conditioned medium prepared by mixing growth medium from a growing Arabidopsis suspension culture 1:4 with fresh medium supplemented with 0.5M mannitol. The protoplasts were then poured into 60 x 15 mm petri dishes and incubated in the dark at 21 - 24 ° C for 21 hours.
Results
Friable, pale yellow callus forms directly from seeds germinated on agar plates containing high auxin levels and grows rapidly to approximately one centimeter in diameter within three weeks (Figure 1A). This callus can be transferred onto fresh agar plates or directly into liquid medium. When inoculated into liquid medium and placed on a gyrotory shaker, the friable dumps of callus slough o f f cells to e s t a b l i s h a c h u n k y p r i m a r y cell s u s p e n s i o n that c o n s i s t s o f b o t h l a r g e a n d s m a l l cell a g g r e g a t e s ( F i g u r e 1B, N e g r u t i u et. al, 1 9 7 5 ) . The smaller clumps of cells r e m o v e d f r o m t h e s e i n i t i a l c u l t u r e s are u s e d as i n o c u l u m for fresh secondary cultures. Within 3-5 weeks (with weekly m e d i u m e x c h a n g e s ) t h e s e c u l t u r e s g r o w to a d e n s i t y o f approximately one gram fresh weight per 24 ml of medium, and contain many small aggregates composed of 8 - 20 c e l l s ( F i g u r e 1C). T h e s e l a t t e r c u l t u r e s a r e t h e n u s e d to p r e p a r e p r o t o p l a s t s ( F i g u r e 1D). R e l a t i v e l y f e w s i n g l e cells are o b s e r v e d i n t h e s e c o n d a r y c u l t u r e s u s e d to g e n e r a t e p r o t o p l a s t s . T h o s e s i n g l e c a l l s t h a t d o o c c u r t e n d to b e large, e l o n g a t e d a n d i r r e g u l a r l y s h a p e d . W e h a v e o b s e r v e d t h a t i n s e c o n d a r y c u l t u r e s m a i n t a i n e d b e y o n d t h r e e to f i v e w e e k s , the a v e r a g e size o f t h e cell a g g r e g a t e s i n c r e a s e s a n d the cultures are no longer a good source of protoplasts. However, the small dumps that invariably remain in these o l d e r c u l t u r e s can b e u s e d to i n o c u l a t e f r e s h cultures. I n this way, w e h a v e m a i n t a i n e d cell l i n e s f o r o v e r o n e year. T h e t r a n s f e c t i o n p r o c e d u r e w e h a v e d e v e l o p e d is similar
Luciferase assays. Lueiferase activity was measured in an Analytical Luminescence Laboratory Monolight 2010 luminometer. Protoplasts were pelleted, resnspended in 200 ul of protoplast lysis buffer (0.1M sodium phosphate pH 7.2 [Fisher Biotech], 5mM DTr [USB]), vortexed vigorously for 5 seconds, and lysed by centrifugation at 1150 x g, 4°C. An aliquot of the supernataut was used directly to measure protein content or was diluted 10-fold with protoplast lysis buffer to assay luciferase activity. Total protein content was measured using the Bradford assay (Bio-Rad) using aeetylated BSA (Promega) as the protein standard. Luciferase assays were performed by mixing 100 ul of 2x assay buffer [60mM Tris-HCI pH 8.0, 20mM MgCI2, 20mM DTr, 2mM EDTA, 2mM ATP (modified from Barnes, 1990)] with 100 ul of diluted protein extract. In another tube, 2x luciferase assay buffer (without ATP) was mixed with an equal volume of 2mM luciferin (Analytical Luminescence Laboratory). Luciferase activity was measured during the initial 10 seconds following injection of 100 el of the lueiferin solution into the protein sample. Luciferase specific activity was calculated as units of activity per microgram of extractable protein. One unit, as calculated by the Monolight 2010, is equal to one-tenth the total number of photons counted multiplied by a factor (0.93) to correct for the efficiency of the detector. For every experiment presented, a control transfection was performed that was identical except that no reporter gene was added. Background luciferase levels in these control samples were then subtracted from each of the samples in which the reporter gene was included.
Fig. 1. Photographs of Arabidopsis tissue and cells at critical points in the establishment of cultures used to generate protoplasts. A) Arabidopsis callus growing on solid medium in a 100 x 25 mm diameter petri dish. B) Arabidopsis suspension culture consisting primarily of visible cell aggregates, growing in a 250 ml flask. C) The same suspension culture cells as in 1B when viewed under a microscope. The size bar represents 60 micrometers. D) Protoplasm generated from suspension eulture ceils. The size bar represents 30 micrometers.
243 to the method described by Damm et. al (1989) to stably transform Arabidopsis mesophyll protoplasts. This method is, in turn, a modification of a procedure developed for tobacco by Negrutiu et. al (1987). Here, we describe the optimization of transient expression of a luciferase reporter gene, pWB216, provided by Dr. Wayne Bames (Washington University School of Medicine). pWB216 is a pBluescribe vector (Stratagene) containing the firefly luciferase gene driven by an enhanced CaMV 35S promoter. This construct is a derivative of the previously described T-DNA construct pWB 146 (Barnes, 1990). Maximal luciferase activity was detected in extracts from protoplasts harvested between 14 and 38 hours following transfection, with only minimal differences detected at these time points (Figure 2). Therefore, in all s u b s e q u e n t experiments, protoplasts were harvested at approximately 21 hours following transfection. The low luciferase activity observed in this experiment relative to other experiments results from using sub-optimal calf thymus carrier DNA (see Figure 3) and, less importantly, 20% PEG instead of 27% PEG during the transfection (.see Figure 4).
107.
10 6tuciferase activity units I ng protein
105•
10 4 • ug c~er ug pWB216
0 50100200 0 50 100200 0 50100200 5 10 20 Treatment
Fig. 3. The effect of calf thymus carrier DNA and reporter plasmid concentration during transfection on the transient expression of luciferase. In this experiment, 1.5 x 106 protoplasts were used in each of three independent replicates performed for each treatment. Average background luciferase levels determined from triplicate control transfections carried out in the absence of pWB216 have been subtracted. The data are plotted as the mean plus or minus the standard deviation.
10 5
Luciferase activity units / ug protein
10 4
10 3 14 24 38 48 Hours
Fig. 2. Effect of incubation time following transfection on transient lueiferase activity. In a volume of 1 ml, 1 x 107 protoplasts were mixed with 100 ug of calf thymus carrier DNA and 37.5 ug pWB216. The PEG concentration was brought to 20% by adding an equal volume of PEG CMS. Protoplasts were then pipetted into separate petri dishes and harvested at the indicated time points. Though six-fold more protoplasts were used than in Figures 3 and 4, the protoplasts constituted a single sample; thus, no error bars are shown. Following lysis of the protoplasts, total luciferase activity and protein yield were determined and the luciferase specific activity calculated. Background luciferase levels obtained from control mock transfections have been subtracted. Note that luciferase activity is plotted on a log scale on this and all subsequent graphs.
In the absense of carrier DNA, transient expression improved steadily as the amount of reporter gene plasmid was increased from 5 ug to 20 ug (Figure 3). However, in the presence of cartier DNA, there was little difference in detectable luciferase levels following transfection with 5, 10, or 20 ug of plasmid. Increased carrier DNA resulted in increased luciferase activity when 5 or 10 micrograms of reporter gene were used, but had little effect when 20 ug of reporter gene plasmid was used. Based on these results, all additional experiments were performed using 10 ug of plasmid DNA and 100 ug of sheared calf thymus DNA. Detectable luciferase specific activity increased more than two-fold as the PEG concentration was raised from 13 to
20%, and increased an additional four-fold between 20 and 27% (Figure 4). Increasing the PEG concentration further to 33% resulted in an insignificant increase in specific activity over the 27% treatment (less than two-fold; note that error bars overlap). However, total extractable protein declined approximately 50% as the PEG was raised from 27% to 33% due to a decline in protoplast survival (data not shown). Clearly, those protoplasts that survived the 33% PEG treatment supported very high levels of luciferase activity, as shown in Figure 4. Nonetheless, we use 27% PEG in our optimized protocol since this treatment allows for high luciferase specific activity coupled with good protoplast survival. 10 8
10 7 Luciferase activity units / ug protein
10 6
10 5 13
20
27
33
% PEG
Fig. 4. The effect of PEG concentration during transfection on transient luciferase expression. For each treatment, protoplast number, reporter gene concentration and calf thymus cartier DNA concentrations were as described in Materials and Methods, while the PEG concentration was varied. Different PEG concentrations were produced by altering the volumes of MaMg and PEG CMS, keeping the final volume constant at 600 ul. Each experiment was done in triplicate both in the presence and absence of l0 ug pWB216 reporter gene plasmid. Background luciferase levels were subtracted as described in Figure 3. The data are plotted as the mean plus or minus the standard deviation.
244 Negrutiu et. al (1987) reported that the Mg +2 ion concentration of the protoplast DNA mixture (before PEG CMS addition) influences transformation efficiency and that the optimum concentration varies from one plant species to another. Therefore, we altered the MaMg solution to test the effects of 15 or 30mM Mg +2 during transfection on detectable luciferase activity. No significant difference in transient luciferase expression was observed (data not shown). Likewise, the metabolism and growth of protoplasts is reportedly stimulated by incubation in conditioned medium (Power and Davy, 1990). In our hands, incubating protoplasts in fresh vs. conditioned medium for the 21 hour period following transfection had no significant effect on luciferase expression (data not shown).
Discussion
To our knowledge, this is the first report describing the optimization of transient gene expression in Arabidopsis. However, Damm et. al (1989) have previously reported a protocol for regenerating stably transformed Arabidopsis plants from protoplasts isolated from leaves, and our method for isolating protoplasts and introducing DNA into them is very similar to theirs. The key to our method is the use of cell suspension cultures as the source of the protoplasts, using culture conditions similar to those described by Negrutiu et. al (1975). The cell suspensions are a reliable source of rapidly growing cells that yield protoplasts which support high levels of transient expression from introduced reporter genes. In our hands, young leaves will also yield excellent protoplasts that support high levels of luciferase transient expression. However, great care must be taken in growing plants for protoplast isolation since the quantity and quality of protoplasts isolated from plants varies greatly from batch to batch, even with plants grown in growth chambers. This is presumably due to intangible environmental or developmental effects. Though we have not completed the optimization of transient expression from leaf protoplasts, the use of MaMg, 120 ug calf thymus carrier DNA, 10 ug pWB216 and 20% PEG with 1.5 x 106 protoplasts in a final volume of 600 ul has worked well. We have also found that protoplasts can be isolated from callus growing on solid medium (Figure 1A). However, the yield of protoplasts from callus is low and the protoplasts do not support efficient expression of an introduced reporter gene. It is important to point out that protoplasts isolated from cell suspension cultures one day are sometimes much more fragile than those isolated on a different day. Survival of fragile protoplasts is better when low PEG concentrations (such as 13%) are used for transfection, but luciferase expression suffers as a result (see Figure 4). Though it is impossible to predict when protoplasts will be fragile, the transformation protocol reported here generally works well. Extractable protein levels are a good indicator for comparing the fragility of different batches of protoplasts, since protein content is proportional to cell number. In our studies involving RNA polymerase I, which require laborious S 1 nuclease protection assays to detect transcripts, we use the
rapid lueiferase assay to test each batch of protoplasts. Protoplasts that support high luciferase expression also support high RNA polymerase I transcription. When we find extractable protein levels and total luciferase levels to be low compared to previous experiments, we know in advance to repeat the experiment with a new batch of protoplasts. The cell suspension culture and protoplast transient expression methods described have a number of beneficial attributes that should prove useful to many researchers studying Arabidopsis. First, the luciferase reporter gene discussed in this paper is expressed from the CaMV 35S promoter, a promoter recognized by RNA polymerase II. As mentioned above, we have found that a transfected Arabidopsis ribosomal RNA gene promoter recognized by RNA polymerase I is also active within the protoplasts (J.H. Doelling and C.S. Pikaard, unpublished) suggesting that our transient expression method will be useful for studies of all three classes of nuclear genes. A second attribute is the ability to establish cultures from seed within a few months without long-term selection to establish stable cell lines. Third, the suspension cultures can be grown in 500 ml or larger flasks, allowing for the production of liters of actively growing calls for biochemical studies, thereby circumventing some of the problems due to the small size of Arabidopsis plants. Fourth, Arabidopsis is a suitable host for a number of plant viruses and bacterial pathogens (Susnova and Polak, 1975; Dong et. al, 1991; Whalen et. al, 1991), suggesting the utility of transient protoplast transfection assays for studying cellular aspects of pathogen invasion and replication in Arabidopsis. Acknowledgements.The
authors wish to thank Dr. Wayne Barnes for the generous gift of pWB216. We also thank Dr. David Ho, Dr. Maitrayee Bhattacharyya and Donna Droste for critical reading of the manuscript, and thank the other members of the Pikaard lab for helpful suggestions and comments. This work was supported by grants to C.S.P. from the National Science Foundation (DMB-9018428), The Midwest Plant Biotechnology Consortium, Agrigenetics Company, and the Center for Plant Science and Biotechnology (Missouri Centers for Advanced Technologies Program).
References Barnes W M (1990) Proc Natl Acad Sci (USA) 87:9183 - 9187. Datum B, Willmitzer L (1988) Mol Gen Genet 213:15 - 20. Datum B, Schmidt R, Willmitzer L (1989) Mol Gen Genet 217:6 - 12. Dong X, Mindrinos M, Davis KR, Ausubel FM (1991) The Plant Cell 3: 61 - 72. Golds TJ, Davey MR, Rech EL, Power JB (1990) In: Pollard JW, Walker JM (eds) Methods in Molecular Biology, vol 6, Plant Cell and Tissue Culture. The Humana Press Clifton, New Jersey pp. 341 - 371. Gamborg OL, Miller RA, Ojima K (1968) Exp Cell Res 50:151 - 158. G m m m t I, Roth E, Paule, MR (1982) Nature (London) 296:173 - 174. Meinke DW (1991) The Plant Cell 3 : 8 5 7 - 866. Murashige T, Skoog F (1962) Physiol Plant 15:473 - 497. Negrutiu I, Beeftink F, Jacobs M (1975) Plant Sci Let 5:293 - 304. Negrutiu I, Shillito R, Potrykns I, Biasini G, Sala F (1987) Plant Mol Biol 8:363 - 373. Power JB, Davey MR (1990) In: Pollard JW, Walker JM (eds) Methods in Molecular Biology, vol 6, Plant Cell and Tissue Culture. The Humana Press Clifton, New Jersey pp. 237-259. Reeder RH (1990) TIG 6 : 3 9 0 - 395. Sollner-Webb B, Mougey EB (1991) TIBS 16:58 - 62. Susnova V, Polak Z (1975) Biol Plant 17:156 - 158. Whalen MC, Innes RW, Bent AF, Staskawicz BJ (1991) The Plant Cell 3: 4 9 - 59.
Plant Cell Reports (1993) t2:241 244
© Springer-Verlag1993
Transient expression in Arabidopsis thaliana protoplasts derived from rapidly established cell suspension cultures Jed H. Doelling and Craig S. Pikaard Biology Department and Center for Plant Science and Biotechnology, Box 1137, Washington University, One Brookings Drive, St. Louis, MO 63130, USA Received August 14, 1992/Revised version received December 14, 1992 - Communicated by J. M. Widholm
Abstract. Arabidopsis thaliana has emerged as a model species for the analysis of genes controlling plant development. However, its small size has impaired biochemical analyses, and the absence of a transient expression system has hampered promoter analysis. Here, we report a method for rapidly establishing A. thaliana suspension cultures that yield protoplasts that can be readily transfected. We have optimized transient expression conditions using a modified polyethylene glycol / calcium nitrate transformation protocol and a Cauliflower Mosaic Virus 35S promoter-luciferase reporter gene construct. Our methods permit isolation of large quantities of rapidly growing cells and analysis of Arabidopsis promoters in vivo in a homologous system. Abbreviations: CaMV, Cauliflower Mosaic Virus; 2,4D, 2,4-dichlorophenoxyacetic acid; MES, 2-(Nmorpholino)ethanesulfonic acid; PEG, polyethylene glycol.
Introduction
Arabidopsis thaliana is rapidly becoming the plant species of choice for molecular genetic studies of fundamental mechanisms controlling plant development. A large number of interesting mutations affecting such things as flower morphology, embryo development, and meristematic cell fate have been characterized (for review see Meinke, 1991). In many cases, the genes responsible for the mutant phenotypes have also been isolated and characterized. Studies of the expression of these genes, once isolated, has been done primarily in stably transformed Arabidopsis plants or in other easily transformed plants, such as tobacco. Production of transgenic plants is time consuming, and while necessary to evaluate tissue specificity and developmental regulation, is an inefficient means of analyzing cis-acting promoter elements recognized by the general transcriptional machinery. Transient expression of genes in plant protoplasts or in microprojectile bombarded intact cells is a much faster way Correspondence to: C. S. Pikaard
of analyzing these essential cis-acting sequences and has been widely used in established plant systems such as tobacco (Golds et. al, 1990). Our laboratory studies transcriptional regulation of the Arabidopsis ribosomal RNA genes, which are transcribed by RNA polymerase I. In animals, RNA polymerase I transcription is highly species-specific, such that the ribosomal genes of one species are generally not expressed in a transcription extract made from another species (Grummt et. al, 1982; reviewed by: Reeder, 1990; SollnerWebb and Mougey, 1991). Our preliminary results suggest that species-specificity of ribosomal gene transcription also occurs in plants (J.H. Doelling and C.S. Pikaard, unpublished). Consequently, we needed to develop a system for the transient expression of Arabidopsis genes in Arabidopsis cells. In this paper, we describe a tissue culture system for A. thaliana "Columbia" from which protoplasts are readily generated. These protoplasts support efficient transient expression of a luciferase reporter gene driven by the CaMV 35S promoter. We present data describing the optimization of several critical parameters for the transient expression system.
Materials and methods Establishment of cell cultures. Arabidopsis thaliana "Columbia" seeds (kindly provided by Dr. Elliot Meyerowitz) were sterilized by immersion in 95% ethanol in a 1.5 ml microfuge tube for 3-5 minutes, followed by immersion in 2.6% sodium hypochlorite solution for 30 minutes with occasional agitation. In a laminar flow hood, seeds were rinsed four times with sterile water and spread onto 0.8% agar plates containing 1X MS salts (Murashige and Skoog, 1962), 1X Gamborg vitamins (Gamborg et. al, 1968), 2 mg/1 2,4D, 0.05 mg/1 kinetin, and 2% glucose (all purchased from Sigma unless otherwise noted) or placed directly in liquid medium of the same composition excluding the agar. Plates were incubated in the light in a growth chamber with 16 hour, 24°C days and 8 hour, 21°C nights. Callus cultures were maintained by transferring the callus to fresh plates approximately once every three weeks. Liquid suspension cultures were initiated from about 300 mg of friable callus (fresh weight) inoculated into 24 ml of liquid medium in 125 ml Erlenmeyer flasks wrapped with aluminum foil to exclude light, Liquid cultures were grown on a gyrotory shaker at 85 rpm using the same temperature regime as the callus cultures. Optimal growth was obtained in a medium containing 3raM MES (USB), 2X Gamborg salts (Gamborg
242 et. al, 1968), 2X Gamborg vitamins, 1 mg/I 2,4D, 0.05 mg/1 Idnetin (hormones were filter-sterilized and added following autoclaving of the medium), and 2% sucrose (Fisher Biotech). The medium was adjusted to pH 5.7 with potassium hydroxide prior to autoclaving. Dense initial cultures were established from the dividing cell aggregates by replacing the old medium with fresh medium once a week. The old medium was most easily removed by pipet after first allowing the clumps of cells to settle. Small cell aggregates accumulated within a month and were used to seed multiple secondary cultures by allowing the largest cell clumps to settle, then transferring 10 ml of the cells still in suspension to a sterile, empty flask. After allowing these cells to settle, the old medium was removed and replaced with 24ml of fresh medium. The medium was replaced weekly for 3-4 weeks until the cultures grew to optimal density (one gram, fresh weight, per 24 ml of medium) for protoplast isolation. Protoplast isolation. Three days after the last medium exchange, approximately 2 grams (fresh weight) of cells in 48 ml of growth medium were poured into a deep petri dish (100 x 25 mm) and the medium removed. Fifteen ml. of filter-sterilized enzyme mix [1.1 g mannitol, 40 mg Maeerozyme R-10 (Serva), 150 mg cellulase "Onozuka R-10" (Serva), 70 mg Driselase, 7mM CaCI 2 (Fisher Biotech), and 3raM MES pH 5.7] was added to the cells. Digestion took place overnight in the dark at 21°C with gentle agitation (45 - 50 rpm) on a rotary shaker. Protoplasts were separated from undigested tissue by consecutive filtration through sterile 230 um, 104 um and 38 um stainless steel wire mesh (Small Parts, Inc. P.O. Box 4650 Miami Lakes, FL 33014-0650) into 50 ml conical tubes. Filtrates were centrifuged 10 minutes at room temperature, 26 x g, in a swinging bucket rotor. The protoplasts were washed twice in 0.34M mannitol, 0.07M CaC12, then resuspended in 10 ml 0.5M mannitol (not buffered) and counted in a hemacytometer. Typical protoplast yields were 2.5 x 107 protoplasts per gram fresh weight of suspension culture cells.
Optimal transfection conditions. Protoplasts stored in mannitol were pelleted and resuspended in ice cold MaMg solution (15mM MgCI 2 [FisherBioteeh], 5mM MES, 0.4M maunitol, pH 5.6) at a concentration of 1.5 x 107 protoplasts / ml and placed on ice. All further manipulations were performed on ice. To 100 ul MaMg solution in a 15 ml disposable conical tube, 100 ug of sheared calf thymus carrier DNA and 10 ug of pWB216 reporter gene construct were added and mixed well. One hundred microliters (1.5 x 106) of protoplast suspension was added to each tube using a wide bore pipet tip (sterile, disposable 0.2ml micropipetor tips with approximately 0.5 cm of the tip cut off) and mixed by pipetting. After a 10 minute incubation, 400 ul PEG CMS [40% PEG 8000 (Fisher Bioteeh), 0.4M mannitol, 0.1M Ca(NO3)2] was added to a final PEG concentration of 27%. Following a thirty minute incubation, this solution was diluted stepwise (diluted two-fold at each step) with 7 volumes ice cold W 5 solution (154mM NaCI [Fisherbiotec], 125mM CaCI2, 5mM KCI [USB], 5mM glucose, 1.5mM MES, 0.4M mannitol, pH 5.8). Protoplasts were pelleted at 26 x g, 4°C, and the pellets washed once with a solution consisting of 2 ml 0.5M mannitol and 0.5 ml W 5 solution. Protoplasts were resuspended in 3 ml conditioned medium prepared by mixing growth medium from a growing Arabidopsis suspension culture 1:4 with fresh medium supplemented with 0.5M mannitol. The protoplasts were then poured into 60 x 15 mm petri dishes and incubated in the dark at 21 - 24 ° C for 21 hours.
Results
Friable, pale yellow callus forms directly from seeds germinated on agar plates containing high auxin levels and grows rapidly to approximately one centimeter in diameter within three weeks (Figure 1A). This callus can be transferred onto fresh agar plates or directly into liquid medium. When inoculated into liquid medium and placed on a gyrotory shaker, the friable dumps of callus slough o f f cells to e s t a b l i s h a c h u n k y p r i m a r y cell s u s p e n s i o n that c o n s i s t s o f b o t h l a r g e a n d s m a l l cell a g g r e g a t e s ( F i g u r e 1B, N e g r u t i u et. al, 1 9 7 5 ) . The smaller clumps of cells r e m o v e d f r o m t h e s e i n i t i a l c u l t u r e s are u s e d as i n o c u l u m for fresh secondary cultures. Within 3-5 weeks (with weekly m e d i u m e x c h a n g e s ) t h e s e c u l t u r e s g r o w to a d e n s i t y o f approximately one gram fresh weight per 24 ml of medium, and contain many small aggregates composed of 8 - 20 c e l l s ( F i g u r e 1C). T h e s e l a t t e r c u l t u r e s a r e t h e n u s e d to p r e p a r e p r o t o p l a s t s ( F i g u r e 1D). R e l a t i v e l y f e w s i n g l e cells are o b s e r v e d i n t h e s e c o n d a r y c u l t u r e s u s e d to g e n e r a t e p r o t o p l a s t s . T h o s e s i n g l e c a l l s t h a t d o o c c u r t e n d to b e large, e l o n g a t e d a n d i r r e g u l a r l y s h a p e d . W e h a v e o b s e r v e d t h a t i n s e c o n d a r y c u l t u r e s m a i n t a i n e d b e y o n d t h r e e to f i v e w e e k s , the a v e r a g e size o f t h e cell a g g r e g a t e s i n c r e a s e s a n d the cultures are no longer a good source of protoplasts. However, the small dumps that invariably remain in these o l d e r c u l t u r e s can b e u s e d to i n o c u l a t e f r e s h cultures. I n this way, w e h a v e m a i n t a i n e d cell l i n e s f o r o v e r o n e year. T h e t r a n s f e c t i o n p r o c e d u r e w e h a v e d e v e l o p e d is similar
Luciferase assays. Lueiferase activity was measured in an Analytical Luminescence Laboratory Monolight 2010 luminometer. Protoplasts were pelleted, resnspended in 200 ul of protoplast lysis buffer (0.1M sodium phosphate pH 7.2 [Fisher Biotech], 5mM DTr [USB]), vortexed vigorously for 5 seconds, and lysed by centrifugation at 1150 x g, 4°C. An aliquot of the supernataut was used directly to measure protein content or was diluted 10-fold with protoplast lysis buffer to assay luciferase activity. Total protein content was measured using the Bradford assay (Bio-Rad) using aeetylated BSA (Promega) as the protein standard. Luciferase assays were performed by mixing 100 ul of 2x assay buffer [60mM Tris-HCI pH 8.0, 20mM MgCI2, 20mM DTr, 2mM EDTA, 2mM ATP (modified from Barnes, 1990)] with 100 ul of diluted protein extract. In another tube, 2x luciferase assay buffer (without ATP) was mixed with an equal volume of 2mM luciferin (Analytical Luminescence Laboratory). Luciferase activity was measured during the initial 10 seconds following injection of 100 el of the lueiferin solution into the protein sample. Luciferase specific activity was calculated as units of activity per microgram of extractable protein. One unit, as calculated by the Monolight 2010, is equal to one-tenth the total number of photons counted multiplied by a factor (0.93) to correct for the efficiency of the detector. For every experiment presented, a control transfection was performed that was identical except that no reporter gene was added. Background luciferase levels in these control samples were then subtracted from each of the samples in which the reporter gene was included.
Fig. 1. Photographs of Arabidopsis tissue and cells at critical points in the establishment of cultures used to generate protoplasts. A) Arabidopsis callus growing on solid medium in a 100 x 25 mm diameter petri dish. B) Arabidopsis suspension culture consisting primarily of visible cell aggregates, growing in a 250 ml flask. C) The same suspension culture cells as in 1B when viewed under a microscope. The size bar represents 60 micrometers. D) Protoplasm generated from suspension eulture ceils. The size bar represents 30 micrometers.
243 to the method described by Damm et. al (1989) to stably transform Arabidopsis mesophyll protoplasts. This method is, in turn, a modification of a procedure developed for tobacco by Negrutiu et. al (1987). Here, we describe the optimization of transient expression of a luciferase reporter gene, pWB216, provided by Dr. Wayne Bames (Washington University School of Medicine). pWB216 is a pBluescribe vector (Stratagene) containing the firefly luciferase gene driven by an enhanced CaMV 35S promoter. This construct is a derivative of the previously described T-DNA construct pWB 146 (Barnes, 1990). Maximal luciferase activity was detected in extracts from protoplasts harvested between 14 and 38 hours following transfection, with only minimal differences detected at these time points (Figure 2). Therefore, in all s u b s e q u e n t experiments, protoplasts were harvested at approximately 21 hours following transfection. The low luciferase activity observed in this experiment relative to other experiments results from using sub-optimal calf thymus carrier DNA (see Figure 3) and, less importantly, 20% PEG instead of 27% PEG during the transfection (.see Figure 4).
107.
10 6tuciferase activity units I ng protein
105•
10 4 • ug c~er ug pWB216
0 50100200 0 50 100200 0 50100200 5 10 20 Treatment
Fig. 3. The effect of calf thymus carrier DNA and reporter plasmid concentration during transfection on the transient expression of luciferase. In this experiment, 1.5 x 106 protoplasts were used in each of three independent replicates performed for each treatment. Average background luciferase levels determined from triplicate control transfections carried out in the absence of pWB216 have been subtracted. The data are plotted as the mean plus or minus the standard deviation.
10 5
Luciferase activity units / ug protein
10 4
10 3 14 24 38 48 Hours
Fig. 2. Effect of incubation time following transfection on transient lueiferase activity. In a volume of 1 ml, 1 x 107 protoplasts were mixed with 100 ug of calf thymus carrier DNA and 37.5 ug pWB216. The PEG concentration was brought to 20% by adding an equal volume of PEG CMS. Protoplasts were then pipetted into separate petri dishes and harvested at the indicated time points. Though six-fold more protoplasts were used than in Figures 3 and 4, the protoplasts constituted a single sample; thus, no error bars are shown. Following lysis of the protoplasts, total luciferase activity and protein yield were determined and the luciferase specific activity calculated. Background luciferase levels obtained from control mock transfections have been subtracted. Note that luciferase activity is plotted on a log scale on this and all subsequent graphs.
In the absense of carrier DNA, transient expression improved steadily as the amount of reporter gene plasmid was increased from 5 ug to 20 ug (Figure 3). However, in the presence of cartier DNA, there was little difference in detectable luciferase levels following transfection with 5, 10, or 20 ug of plasmid. Increased carrier DNA resulted in increased luciferase activity when 5 or 10 micrograms of reporter gene were used, but had little effect when 20 ug of reporter gene plasmid was used. Based on these results, all additional experiments were performed using 10 ug of plasmid DNA and 100 ug of sheared calf thymus DNA. Detectable luciferase specific activity increased more than two-fold as the PEG concentration was raised from 13 to
20%, and increased an additional four-fold between 20 and 27% (Figure 4). Increasing the PEG concentration further to 33% resulted in an insignificant increase in specific activity over the 27% treatment (less than two-fold; note that error bars overlap). However, total extractable protein declined approximately 50% as the PEG was raised from 27% to 33% due to a decline in protoplast survival (data not shown). Clearly, those protoplasts that survived the 33% PEG treatment supported very high levels of luciferase activity, as shown in Figure 4. Nonetheless, we use 27% PEG in our optimized protocol since this treatment allows for high luciferase specific activity coupled with good protoplast survival. 10 8
10 7 Luciferase activity units / ug protein
10 6
10 5 13
20
27
33
% PEG
Fig. 4. The effect of PEG concentration during transfection on transient luciferase expression. For each treatment, protoplast number, reporter gene concentration and calf thymus cartier DNA concentrations were as described in Materials and Methods, while the PEG concentration was varied. Different PEG concentrations were produced by altering the volumes of MaMg and PEG CMS, keeping the final volume constant at 600 ul. Each experiment was done in triplicate both in the presence and absence of l0 ug pWB216 reporter gene plasmid. Background luciferase levels were subtracted as described in Figure 3. The data are plotted as the mean plus or minus the standard deviation.
244 Negrutiu et. al (1987) reported that the Mg +2 ion concentration of the protoplast DNA mixture (before PEG CMS addition) influences transformation efficiency and that the optimum concentration varies from one plant species to another. Therefore, we altered the MaMg solution to test the effects of 15 or 30mM Mg +2 during transfection on detectable luciferase activity. No significant difference in transient luciferase expression was observed (data not shown). Likewise, the metabolism and growth of protoplasts is reportedly stimulated by incubation in conditioned medium (Power and Davy, 1990). In our hands, incubating protoplasts in fresh vs. conditioned medium for the 21 hour period following transfection had no significant effect on luciferase expression (data not shown).
Discussion
To our knowledge, this is the first report describing the optimization of transient gene expression in Arabidopsis. However, Damm et. al (1989) have previously reported a protocol for regenerating stably transformed Arabidopsis plants from protoplasts isolated from leaves, and our method for isolating protoplasts and introducing DNA into them is very similar to theirs. The key to our method is the use of cell suspension cultures as the source of the protoplasts, using culture conditions similar to those described by Negrutiu et. al (1975). The cell suspensions are a reliable source of rapidly growing cells that yield protoplasts which support high levels of transient expression from introduced reporter genes. In our hands, young leaves will also yield excellent protoplasts that support high levels of luciferase transient expression. However, great care must be taken in growing plants for protoplast isolation since the quantity and quality of protoplasts isolated from plants varies greatly from batch to batch, even with plants grown in growth chambers. This is presumably due to intangible environmental or developmental effects. Though we have not completed the optimization of transient expression from leaf protoplasts, the use of MaMg, 120 ug calf thymus carrier DNA, 10 ug pWB216 and 20% PEG with 1.5 x 106 protoplasts in a final volume of 600 ul has worked well. We have also found that protoplasts can be isolated from callus growing on solid medium (Figure 1A). However, the yield of protoplasts from callus is low and the protoplasts do not support efficient expression of an introduced reporter gene. It is important to point out that protoplasts isolated from cell suspension cultures one day are sometimes much more fragile than those isolated on a different day. Survival of fragile protoplasts is better when low PEG concentrations (such as 13%) are used for transfection, but luciferase expression suffers as a result (see Figure 4). Though it is impossible to predict when protoplasts will be fragile, the transformation protocol reported here generally works well. Extractable protein levels are a good indicator for comparing the fragility of different batches of protoplasts, since protein content is proportional to cell number. In our studies involving RNA polymerase I, which require laborious S 1 nuclease protection assays to detect transcripts, we use the
rapid lueiferase assay to test each batch of protoplasts. Protoplasts that support high luciferase expression also support high RNA polymerase I transcription. When we find extractable protein levels and total luciferase levels to be low compared to previous experiments, we know in advance to repeat the experiment with a new batch of protoplasts. The cell suspension culture and protoplast transient expression methods described have a number of beneficial attributes that should prove useful to many researchers studying Arabidopsis. First, the luciferase reporter gene discussed in this paper is expressed from the CaMV 35S promoter, a promoter recognized by RNA polymerase II. As mentioned above, we have found that a transfected Arabidopsis ribosomal RNA gene promoter recognized by RNA polymerase I is also active within the protoplasts (J.H. Doelling and C.S. Pikaard, unpublished) suggesting that our transient expression method will be useful for studies of all three classes of nuclear genes. A second attribute is the ability to establish cultures from seed within a few months without long-term selection to establish stable cell lines. Third, the suspension cultures can be grown in 500 ml or larger flasks, allowing for the production of liters of actively growing calls for biochemical studies, thereby circumventing some of the problems due to the small size of Arabidopsis plants. Fourth, Arabidopsis is a suitable host for a number of plant viruses and bacterial pathogens (Susnova and Polak, 1975; Dong et. al, 1991; Whalen et. al, 1991), suggesting the utility of transient protoplast transfection assays for studying cellular aspects of pathogen invasion and replication in Arabidopsis. Acknowledgements.The
authors wish to thank Dr. Wayne Barnes for the generous gift of pWB216. We also thank Dr. David Ho, Dr. Maitrayee Bhattacharyya and Donna Droste for critical reading of the manuscript, and thank the other members of the Pikaard lab for helpful suggestions and comments. This work was supported by grants to C.S.P. from the National Science Foundation (DMB-9018428), The Midwest Plant Biotechnology Consortium, Agrigenetics Company, and the Center for Plant Science and Biotechnology (Missouri Centers for Advanced Technologies Program).
References Barnes W M (1990) Proc Natl Acad Sci (USA) 87:9183 - 9187. Datum B, Willmitzer L (1988) Mol Gen Genet 213:15 - 20. Datum B, Schmidt R, Willmitzer L (1989) Mol Gen Genet 217:6 - 12. Dong X, Mindrinos M, Davis KR, Ausubel FM (1991) The Plant Cell 3: 61 - 72. Golds TJ, Davey MR, Rech EL, Power JB (1990) In: Pollard JW, Walker JM (eds) Methods in Molecular Biology, vol 6, Plant Cell and Tissue Culture. The Humana Press Clifton, New Jersey pp. 341 - 371. Gamborg OL, Miller RA, Ojima K (1968) Exp Cell Res 50:151 - 158. G m m m t I, Roth E, Paule, MR (1982) Nature (London) 296:173 - 174. Meinke DW (1991) The Plant Cell 3 : 8 5 7 - 866. Murashige T, Skoog F (1962) Physiol Plant 15:473 - 497. Negrutiu I, Beeftink F, Jacobs M (1975) Plant Sci Let 5:293 - 304. Negrutiu I, Shillito R, Potrykns I, Biasini G, Sala F (1987) Plant Mol Biol 8:363 - 373. Power JB, Davey MR (1990) In: Pollard JW, Walker JM (eds) Methods in Molecular Biology, vol 6, Plant Cell and Tissue Culture. The Humana Press Clifton, New Jersey pp. 237-259. Reeder RH (1990) TIG 6 : 3 9 0 - 395. Sollner-Webb B, Mougey EB (1991) TIBS 16:58 - 62. Susnova V, Polak Z (1975) Biol Plant 17:156 - 158. Whalen MC, Innes RW, Bent AF, Staskawicz BJ (1991) The Plant Cell 3: 4 9 - 59.
