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First published online June 4, 2004; 10.1104/pp.104.040121 Plant Physiology 135:916-926 (2004) © 2004 American Society of Plant Biologists Intracellular Localization of Arabidopsis Sulfurtransferases1Institute for Botany (M.B., J.P.) and Institute for Plant Diseases and Plant Protection (C.D.), University of Hannover, D–30419 Hannover, Germany; Institute for Plant Biology, Technical University of Braunschweig, D–38106, Braunschweig, Germany (K.N.); and Laboratory of Ultrastructures, INTA University of Chile, 5540 Macul Santiago, Chile (W.D.S.)
Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism.
All members in the sulfurtransferase (Str)/rhodanese protein family in archaea, eubacteria, and eukaryota are unified by characteristic well-defined sequence domains (Bordo and Bork, 2002  ). These domains are found as tandem repeats, with the C-terminal domain containing the active site Cys residue, as single-domain proteins or as members of multidomain proteins (Bordo and Bork, 2002). The 18 proteins identified in Arabidopsis which contain at least one Str signature were classified into six groups on the basis of their sequence homology (Bauer and Papenbrock, 2002; http://arabidopsis.org/info/genefamily/STR_genefamily.html). Group I consists of two Str proteins with two-domain; the five proteins in group VI contain only the C-terminal Str signature and thus possess similarity to the single-domain Str from bacteria.
Strs catalyze the transfer of a sulfur atom from suitable sulfur donors to a nucleophilic acceptor. Specific biological roles for most members of this superfamily have not been established (Spallarossa et al., 2001
Strs have been identified in different compartments in living organisms. In Escherichia coli, seven Str proteins were identified; a single-domain Str, GlpE, is a cytoplasmic protein, whereas at least one two-domain Str was localized in the periplasm (Ray et al., 2000 Our aim of the research in this paper is the functional analysis of all members of the nuclear encoded Str multiprotein family in Arabidopsis. Knowledge of their intracellular localization is a prerequisite for the determination of their metabolic roles in multicellular organisms. Computer-based prediction programs are not yet sufficiently reliable and are only indicative of the in vivo localization of proteins. For exclusively immuno-based localization methods, many monospecific antibodies which are also difficult to produce against highly similar proteins would be required. The mitochondrial localization of AtStr1 was demonstrated by several different methods leading to the same conclusions. The transient expression of GFP fusion proteins in Arabidopsis protoplasts was established as a reliable method for the determination of the intracellular localization as confirmed by a number of controls.
Intracellular Localization of AtStr1 in Mitochondria Arabidopsis protoplasts were transformed using a transient expression system with AtStr1 and AtStr2 including (wPS) or excluding (woPS) their putative targeting peptide sequences (Fig. 1; for an overview of the constructs see Table I). Colocalization of the AtStr1wPS/pGFP-N fluorescence with MitoTracker Orange fluorescence, a dye that is specifically enriched in mitochondria, in the same protoplasts using a confocal laser scanning microscope (CLSM) resulted in the same picture. A second control for mitochondrial intracellular localization was used: the targeting peptide of the mitochondrial protein, Ser hydroxymethyltransferase, was fused to the GFP protein, resulting in the same image as transient transformation with the AtStr1 fusion construct (data not shown). These results indicated import of the AtStr1 protein into the mitochondria (Fig. 1, A and B). The same localization results were obtained with a fluorescence microscope (Fig. 1D). The corresponding bright field picture visualizes the protoplast's cell membrane, demonstrates the intactness of the protoplasts, and demonstrates the position of the chloroplasts (Fig. 1C). AtStr1 expressed without its putative targeting peptide sequence remained in the cytoplasm (Fig. 1F). The fusion construct of AtStr1 with the targeting peptide sequence and the GFP-encoding cDNA sequence at the 5' end (AtStr1wPS/pGFP-C) resulted in a fluorescence image identical to the transformed pGFP-C vector alone (data not shown). This indicates that the N-terminal targeting peptide is recognized by the import machinery of the mitochondria.
AtStr2 protein was described to be cytoplasmic (Hatzfeld and Saito, 2000 ; Nakamura et al., 2000). However, the 5' end of the AtStr2 sequence was possibly not correctly determined, since the EST clone ATTS6033 obtained from the Arabidopsis Biological Resource Center, Ohio State University, contained a putative targeting peptide of 48 amino acids (Table II; Papenbrock and Schmidt, 2000b). Therefore protoplasts were transformed with the respective constructs including or excluding the 5' extension (AtStr2wPS/pGFP-N, AtStr2wPS/pGFP-C, and AtStr2woPS/pGFP-N). All AtStr2 fusion proteins remained in the cytoplasm. Obviously, the additional DNA at the 5' end did not encode a putative targeting peptide (Fig. 1, G–J; AtStr2wPS/pGFP-C localization data not shown).
To demonstrate the reliability of localization studies using GFP fusion proteins, methods based on antibody specificity were applied. Mitochondria were purified from Arabidopsis cell cultures, chloroplasts were enriched from green Arabidopsis plants, and total protein extracts were prepared from green Arabidopsis plants. In the western-blot analysis, a protein of about 35 kD was detected in the mitochondrial fraction, corresponding to the predicted size of the mature AtStr1 protein (35.6 kD; Fig. 2A). In total extracts, a protein of about 43 kD was found close to the predicted size of the AtStr1 protein including the targeting peptide (41.9 kD).
The proteome of mitochondria purified from Arabidopsis cell cultures was separated by two-dimensional gel electrophoresis (50 µg protein) and was subject to western-blot analysis (Fig. 2B, top). In comparison to the western blot of the one-dimensional gel, the development of the color reaction was prolonged to detect as many cross-reacting proteins as possible without obtaining too much background. Two protein spots, a major and a minor, were identified in the immunoreaction. The molecular mass and the pI of the major spot are in agreement with the predicted values (35.6 kD/pI 5.01) of the mature AtStr1 protein. In parallel, the mitochondrial proteome (1 mg protein) was separated under the same conditions, and the gel was Coomassie-stained. The protein spots corresponding to the proteins recognized by the AtStr1-specific antibody are marked by arrows (Fig. 2B, bottom). In the MALDI/TOF-MS and ESI-MS/MS analysis, both the major and the minor protein spots were identified as AtStr1 proteins. The difference in the molecular mass may be explained by a posttranslational modification, such as a phosphorylation. The nature of the modification is still unknown. The sequence of the N-terminal peptide of the mature AtStr1 could be determined, and the computer-predicted cleavage site was confirmed experimentally. The total length of targeting peptide was calculated to 58 amino acids; the bar marks the cleavage site: MASTL...WARRA_MASTG.
Group VI of the AtStr family contains five members which have single-domains (Table II). To clarify their function, as many features as possible of each single AtStr should be determined. Intracellular localization of four of the single-domain AtStrs was analyzed. To date, no EST clone of AtStr17 has been isolated (http://www.uni-frankfurt.de/fb15/botanik/mcb/AFGN/papen.htm). RT-PCR using different mRNA-pools and screening of cDNA libraries with sequence specific primers was not successful, indicating that the gene encoding AtStr17 may be a pseudogene. Nicotiana tabacum leaf cells were transformed with the transient expression system with the fusion constructs AtStr14/pGFP-N and AtStr14/pGFP-C or AtStr18/pGFP-N and AtStr18/pGFP-C using the ballistic method of particle gun bombardment. Transformed tobacco leaf epidermal and guard cells were analyzed by fluorescence microscopy after overnight incubation (Fig. 3). AtStr14 was transported into plastids as seen in the images of the GFP fluorescence in plastids of epidermal and guard cells (Fig. 3, B and C). The bright field image of an epidermal N. tabacum cell shows the cell dimensions (Fig. 3A). In pretests, leaves of Arabidopsis were also transformed by particle bombardment and led to the same localization of the Arabidopsis proteins as the heterologous transformation of N. tabacum leaves. Controls using a GFP fusion construct containing the transit peptide of Rubisco revealed comparable images, as shown in Figure 3, B and C (data not shown). Figure 3, D and E, show the same guard cell transformed with the AtStr18/pGFP-N construct: GFP fluorescence was detected in the cytoplasm.
To examine targeting of four out of five of the single-domain AtStr, fusion constructs of AtStr14, AtStr15, AtStr16, and AtStr18 with pGFP-N or pGFP-C were introduced into Arabidopsis protoplasts, incubated overnight at room temperature, and visualized by fluorescence microscopy (Fig. 4). Bright field images were taken to visualize the protoplast's cell membrane and chloroplasts. AtStr14, AtStr15, and AtStr16 were localized in chloroplasts, whereas AtStr18 remained in the cytoplasm. The choice of the transformation method, either particle bombardment or polyethylene glycol-mediated protoplast transformation, did not affect the localization results as shown by Figures 3 and 4.
The AtStr15 Protein Is Associated with the Thylakoid Membrane In contrast to AtStr14 and AtStr16, which were evenly distributed in the chloroplasts which is indicative for soluble proteins, the intracellular localization of the AtStr15 protein was unusual (Fig. 4). It was not possible to unambiguously determine whether the protein was outside of the chloroplast, on the chloroplast envelope, or attached to the thylakoid membranes inside the chloroplast. Therefore we investigated its localization in more detail. Protoplasts were transiently transformed with AtStr15/pGFP-N and analyzed with the CLSM (Fig. 5). In Figure 5C, the merged image of GFP fluorescence and chlorophyll autofluorescence of the same protoplast is shown. In Figure 5D, coordinating lines show the localization of AtStr15 (shown on the sidelines) in more detail. A higher magnification of the same protoplast enabled visualization of the thylakoid membranes and indicated an association of AtStr15 with the thylakoid membrane (Fig. 5, E and F). However, the limited resolution of the CLSM and the extensive emission of the GFP fluorescence made a final conclusion with respect to the subcellular localization of AtStr15 difficult.
Transmission electron immunomicroscopy was used to investigate the subcellular localization further. Protoplasts were transformed with AtStr15/pGFP-N using a transient expression system and then incubated with antibodies directed against GFP, and the secondary antibody was labeled with gold particles. The transmission images show an association of AtStr15 protein with the thylakoid membranes and to a smaller extent localization in the stroma of the chloroplasts (Fig. 6).
The statistical analysis of the density of gold labeling in the chloroplasts indicates an effective transformation. In 43 mitochondria, only a total of 3 gold particles were found; in the profiles of the plasma membrane from 10 protoplasts just 4 gold particles were detected. In an area of 152 µm–2 of cytoplasm, 38 gold particles were counted. In 27 chloroplasts, with an area of 144 µm–2, 234 gold particles were found. More than 74% of the gold label in chloroplast was associated with the thylakoid membranes, the remaining in stroma (Table III). As controls for the specificity of the attachment, sections were incubated either in the absence of the GFP-specific antibody or with preimmune serum. Less than 1 gold particle per 10 µm–2 was seen in these control sections. Only a few areas around some vacuoles displayed 2 or 3 gold particles above this background. However, this compartment is known as having relatively high unspecific affinity.
Computer-Based Prediction of Protein Localization Compared with Experimental Results Several computer programs using different algorithms for the prediction of the intracellular localization of proteins were applied. The results of the prediction by computer programs are summarized in Table II. For three out of six proteins, the localization predictions were in agreement with the experimental results (AtStr1, AtStr14, and AtStr15). For these proteins the probabilities given by both programs PSORT and TargetP were very high. However, results in TargetP for AtStr1 suggested a dual-targeting into mitochondria and chloroplasts. The prediction probabilities for the other three proteins gave lower values, except for a chloroplast stroma localization of AtStr18 in PSORT. In the case of these lower certainties for the prediction, the experimental data gave completely different results.
The Targeting Peptide of the Nuclear Encoded AtStr1 Protein Is Cleaved after Being Imported into Mitochondria
All Str proteins identified in Arabidopsis are nuclear-encoded proteins (Bauer and Papenbrock, 2002
The western-blot analyses of organelle and total protein extracts showed a mitochondrial localization of the mature AtStr1 protein. To identify the correct cleavage site, mitochondria were isolated from suspension cell cultures, the mitochondrial proteome was separated by two-dimensional gel electrophoresis, and AtStr1 was detected by using a monospecific antibody against the protein. The determined size and pI corresponded with the computer prediction. The sequence of the mature protein's N terminus, and therefore the cleavage site, matched the predictions. In this particular case, all prerequisites for a successful application of this method were fulfilled. For a wide-spread use there are limitations, such as the availability of monospecific antibodies. In addition, the isolation of pure organelles from cell cultures is a compromise since the correct localization of proteins might be affected by the intactness of the whole plant organism. However, the isolation of mitochondria (Hausmann et al., 2003
Recently a high throughput analysis of the Arabidopsis mitochondrial proteome derived by liquid chromatography-tandem mass spectrometry was reported (Heazlewood et al., 2004
The transient transformation of Arabidopsis protoplasts using GFP fusion constructs was successfully established in our laboratory as described above. To confirm the translocation results obtained by this method, intact cells of N. tabacum and Arabidopsis leaves, respectively, were additionally transiently transformed by particle bombardment with two single-domain AtStrs. It was reported that the translocation of proteins might be species-dependent. A homologous system was recommended, at least for import experiments of dual-targeting proteins (Lister et al., 2001
It has been speculated that Str proteins are involved in iron-sulfur cluster biosynthesis in mitochondria and plastids as a donor of reduced sulfur (Bonomi et al., 1977
The AtStr15 protein localization data was unusual: it might be associated with the chloroplast membrane or localized inside the chloroplasts (Fig. 4D). Recently, the intracellular localization of a N. tabacum protein, Ntdin, with 56.8% sequence identity to AtStr15 on the amino acid level was exclusively found in chloroplasts (Yang et al., 2003
The resolution in cell images visualized by fluorescence microscopy is sufficient for the localization of soluble organellar proteins and can be merged with the autofluorescence images. However, the investigation of subcellular detail can be drastically improved by the ability to show single nm sections of the cell by CLSM, compared to the fluorescence microscope in which the fluorescence of the whole protoplast is collected. The highly magnified CLSM images of cells transformed with AtStr15 (Fig. 5, E and F) were the grounds for assuming an association with the thylakoid membrane. The images of immunogold labeling by transmission electron microscopy and their statistical evaluation support a preferential association of the AtStr15 molecules to the thylakoid membranes inside the chloroplast. The AtStr15 C terminus contains a hydrophobic region of about 20 amino acids, which is predicted to be transmembrane or membrane-associated (Transmembrane Hidden Markov Model; http://www.expasy.ch/tools). Therefore we assume that the protein is attached to the thylakoid membrane by the C terminus. Studies on a peroxiredoxin protein indicated that the decameric form was attached to the thylakoid membrane, and this depended on the physiological status of the cell (Konig et al., 2002
The computer-based localization prediction for all AtStrs gave putative localizations in the cytoplasm, chloroplast stroma and thylakoid membrane, mitochondrion, peroxisome, and nucleus (Table II). Our experimental approach revealed that the intracellular computer localization predictions were correct for only three out of six proteins. In a study by Millar et al. (2001)
The prediction programs use different algorithms. PSORT is based on an expert system with a knowledge-base and is a collection of "if-then"-type rules (Nakai and Kanehisa, 1992
The members of the multiprotein family of Arabidopsis Str investigated are localized in the cytoplasm, in the mitochondrion, and in plastids. The mitochondrial localization of AtStr1 was shown by western-blot analysis and transient expression of GFP fusion constructs. The transient transformation of either leaf cells or protoplasts with GFP fusion constructs gave similar results. Three of the single-domain AtStr are translocated to plastids, and AtStr15 is closely associated with the thylakoid membrane. The prediction of localization using several computer programs based on different algorithms is correct only to about 50%. Therefore biochemical analysis of the intracellular localization of individual proteins is still required in the age of high-throughput methods and bioinformatics.
Plant Material
Arabidopsis Heynh. (ecotype C24) and Nicotiana tabacum L. (cv Samsun NN) plants were grown on soil in the greenhouse in a 16 h light/8 h dark cycle at a temperature of 23°C/21°C. When necessary, additional light was switched on for 16 h per day to obtain a constant quantum fluence rate of 300 µmol m–2 s–1 (sodium vapor lamps, SON-T Agro 400, Philips, Eindhoven, The Netherlands). Arabidopsis (ecotype C24) suspension cultures were established and grown as described by May and Leaver (1993)
The origins of templates and conditions for the amplification of cDNAs encoding AtStr1 (At1g79230), AtStr2 (At1g16460), AtStr15 (At4g35770), AtStr16 (At5g66040), and AtStr18 (At5g66170) have been described previously (Papenbrock and Schmidt, 2000a The PCR contained 0.2 mM dNTPs (Roth, Karlsruhe, Germany), 0.4 µM of each primer (MWG, Ebersberg, Germany), 1 mM MgCl2 (final concentration, respectively), 0.75 µL RedTaq DNA-Polymerase (Sigma, Taufkirchen, Germany), and about 1 µg template DNA in a final volume of 50 µL. Before starting the first PCR cycle, the DNA was denatured for 180 s at 94°C, followed by 28 PCR cycles of 45 s at 94°C, 45 s at 55°C, and 45 s at 72°C each. The process was finished with an elongation phase of 420 s at 72°C. The amplified PCR fragments were ligated into pBSK-based enhanced GFP containing vectors to obtain either GFP fusions with the 5' end of the GFP coding sequence (pGFP-N) or with the 3' end (pGFP-C) and were introduced into the Escherichia coli strain XL1-blue. The gene-cassettes were driven by the CaMV-35S promoter with a double enhancer and the polyA-tail from CaMV-35S. Clones were sequenced for conformation of the insert using specific primers GFPforward 5'-CTGGAGTTCGTGACCGCCGCCGG-3' or GFPreverse 5'-GCTTGCCGTAGGTGGCATCGCCCT-3' (MWG Biotech, Ebersberg, Germany). The expressed Str proteins were either fused with the N terminus of the GFP (pGFP-N) or with the C terminus of the GFP (pGFP-C).
Plasmid DNA from the constructs described above was column-purified (Plasmid Midi Kit, Qiagen, Hilden, Germany). Gold particles (1.0 µm gold microcarrier, Bio-Rad Laboratories GmbH, Munich) were covered with plasmid DNA as follows (protocol for 6 repetitions): 3 mg gold was washed in pure 70% ethanol, centrifuged for 1 min at 1,925g with braking. The gold pellet was resuspended in 50 µL H2O and again centrifuged at 47g without braking. The pellet was resuspended in 50 µL 50% glycerol by mixing and ultrasonification for 10 s. After adding 7 µg plasmid DNA dissolved in 7 µL H2O and incubation for 5 min on ice, 50 µL 2.5 M CaCl2 and 20 µL cold 1 M spermidine were added. The suspension was centrifuged for 15 s at 47g and the pellet was resuspended in 100% ethanol by mixing and ultrasonification. For the bombardment, 5 µL of the DNA-covered gold particles were distributed on a macrocarrier/flying disc (Bio-Rad). The rupture discs (Bio-Rad) used could withstand 900 pounds per square inch. Pieces (4 x 4 cm) of fully expanded N. tabacum leaves were cut from 4-week-old plants by avoiding the middle rib. The leaf pieces were placed upside down on moistened filter paper in petri dishes (9 cm diameter). The particle delivery system was used according to the manufacturer's instructions (Bio-Rad). The treated leaf pieces were incubated for about 16 h at room temperature. Then the epidermis of the tobacco leaves was removed and placed in 0.3 M sorbitol on a glass slide for microscopic analysis.
The younger rosette leaves of 3-week-old Arabidopsis plants were used for the preparation of protoplasts according to Damm et al. (1989) In some experiments MitoTracker Orange CMTMRos (Molecular Probes, Eugene, OR) was used to stain mitochondria. The dye was dissolved in dimethyl sulfoxide to 1 mM and was incubated in a final concentration of 0.5 µM with the protoplast suspension for 15 min.
The transiently transformed cells and protoplasts were analyzed with an Axioskop microscope (Carl Zeiss, Jena, Germany). The GFP fluorescence was collected with the band pass filter (BP 450–490) for excitation and with the long pass filter (LP 520) for emission. The generation of transmission micrographs for visualization of nonfluorescent protoplast structures was achieved using the manufacturer's filter settings. Confocal imaging of transformed protoplasts and stained mitochondria was performed with the True Confocal Scanner (Leica, TCS SP2, Solms, Germany). Specimens were examined using Leica 20x and 63x water immersion objectives. GFP fluorescence was excited with the argon laser (488 nm) and detected at 515 nm to 520 nm. MitoTracker Orange CMTMRos was excited with the green helium neon laser (543 nm) and detected at 575 nm to 585 nm. Far-red autofluorescence of chlorophyll was detected at 650 nm to 670 nm. All images were edited with Corel Photo Paint 10.
The suspension of protoplasts was centrifuged at 375g in an Eppendorf microtube; the sediment was layered with 10 uL 1.5% gelatin in medium II and was then cooled on ice. After solidification of the gelatin, the samples were covered with 1 mL of fixative, consisting of 4% (w/v) freshly depolymerized paraformaldehyde prepared in medium II and containing 0.5% (w/v) glutaraldehyde. After fixation for 30 min at 4°C, the samples were washed with medium II, dehydrated at low temperature with graded alcohols, and embedded in LR-Gold as described previously (Sierralta, 2001
Mitochondria were isolated from Arabidopsis suspension cultures as described by Kruft et al. (2001)
The analyses of DNA and amino acid sequences were performed with the programs EditSeq and MapDraw in DNASTAR (Madison, WI). For the computer-based prediction of the protein localization, different programs were applied (mainly PSORT, SignalP V2.0, TargetP, and further programs in http://www.expasy.ch/tools). After the transient transformation methods had been established, the transformations were performed with each clone at least five times, resulting always in the same intracellular localization.
We thank P. von Trzebiatowski for her excellent technical assistance. We thank Prof. H.P. Braun and H. Eubel, Hannover, Germany, for carrying out the two-dimensional gel electrophoresis and the western blot. We appreciate the mass spectrometry analysis of Str1 done by Dr. C. Lemaitre-Guillier and Prof. A. Van Dorsselaer, Strasbourg, France. We are grateful to A. Wachter and Prof. T. Rausch (Heidelberg) for the generous donation of GFP control constructs for mitochondrial, peroxisomal, and plastidal localization initially used. Many thanks to Dr. R. Hänsch (Braunschweig, Germany) for his initial help in particle bombardment experiments. In our laboratory, particle bombardment experiments were established by M. Brusch. Prof. Dr. W.D. Sierralta visited Hannover in the frame of the AleChile Academic Exchange Program supported by DAAD and Conicyt (project no. 2001/061). We thank Dr. M. Hawkesford, Rothamsted Research, United Kingdom, for correcting the English language of the manuscript. Received January 31, 2004; returned for revision March 16, 2004; accepted March 16, 2004.
1 This work was supported by the Deutsche Forschungsgemeinschaft (projects PA764/1–3 to M.B. and J.P. and ME1266/14–2 to K.N) and by an EU funded research consortium in the VRTP-IMPACT project (C.D.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.040121. * Corresponding author; e-mail jutta.papenbrock{at}botanik.uni-hannover.de; fax 49–511–762–3992.
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ABSTRACT
RESULTS
-mercaptoethanol, and 0.1% [w/v] bovine serum albumine). After application of a vacuum for 15 min, the leaves were incubated on a shaker (40 rpm) for 60 min at room temperature. The suspension was filtered through a 75 µm-pore nylon net, distributed on 4 2 mL-tubes, and centrifuged for 2 min at 95g and 4°C. The pellets were washed twice with 500 µL of medium II (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 2 mM MES/KOH, pH 5.7) and finally incubated for 30 min on ice in medium II. After centrifugation for 2 min at 95g and 4°C, the pellets were carefully resuspended in 150 µL of medium III (0.4 M mannitol, 15 mM MgCl2, and 4 mM MES/KOH, pH 5.7). For the transformation, 100 µL of the protoplast suspension was mixed carefully with 15 µg column-purified plasmid DNA and 110 µL medium IV (4 g polyethylene glycol 4,000, 3 mL H2O, 2.5 mL 0.8 M mannitol, and 1 mL 1 M CaCl2), and incubated for 30 min at 23°C. To remove the polyethylene glycol, the solution was washed with 1 mL of medium II and centrifuged for 2 min at 95g and 4°C. The pellet was resuspended in 50 µL of medium II and incubated overnight at room temperature. 
