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First published online December 14, 2004; 10.1105/tpc.104.026641 © 2005 American Society of Plant Biologists Arabidopsis POLYOL TRANSPORTER5, a New Member of the Monosaccharide Transporter-Like Superfamily, Mediates H+-Symport of Numerous Substrates, Including myo-Inositol, Glycerol, and Ribose
a Molekulare Pflanzenphysiologie, Universität Erlangen-Nürnberg, D-91058 Erlangen, Germany 1 To whom correspondence should be addressed. E-mail nsauer{at}biologie.uni-erlangen.de; fax 49-09131-85-28751.
Six genes of the Arabidopsis thaliana monosaccharide transporter-like (MST-like) superfamily share significant homology with polyol transporter genes previously identified in plants translocating polyols (mannitol or sorbitol) in their phloem (celery [Apium graveolens], common plantain [Plantago major], or sour cherry [Prunus cerasus]). The physiological role and the functional properties of this group of proteins were unclear in Arabidopsis, which translocates sucrose and small amounts of raffinose rather than polyols. Here, we describe POLYOL TRANSPORTER5 (AtPLT5), the first member of this subgroup of Arabidopsis MST-like transporters. Transient expression of an AtPLT5–green fluorescent protein fusion in plant cells and functional analyses of the AtPLT5 protein in yeast and Xenopus oocytes demonstrate that AtPLT5 is located in the plasma membrane and characterize this protein as a broad-spectrum H+-symporter for linear polyols, such as sorbitol, xylitol, erythritol, or glycerol. Unexpectedly, however, AtPLT5 catalyzes also the transport of the cyclic polyol myo-inositol and of different hexoses and pentoses, including ribose, a sugar that is not transported by any of the previously characterized plant sugar transporters. RT-PCR analyses and AtPLT5 promoter-reporter gene plants revealed that AtPLT5 is most strongly expressed in Arabidopsis roots, but also in the vascular tissue of leaves and in specific floral organs. The potential physiological role of AtPLT5 is discussed.
Linear polyols, such as sorbitol or mannitol, are found in high concentrations in the phloem sap of plants from several families, such as Rosaceae, Apiaceae, or Plantaginaceae (Zimmermann and Ziegler, 1975  ). After their synthesis in leaves by sugar phosphate reductases and polyol phosphate phosphatases, linear polyols are loaded into the phloem by polyol-H+ symporters that accumulate their substrates to concentrations of several hundred millimolar (Zimmermann and Ziegler, 1975; Lohaus and Fischer, 2002; G. Lohaus, unpublished data). cDNAs encoding these transporters have been cloned recently from celery (Apium graveolens, Apiaceae; Noiraud et al., 2001), from sour cherry (Prunus cerasus, Rosaceae; Gao et al., 2003), and from common plantain (Plantago major, Plantaginaceae; Ramsperger-Gleixner et al., 2004). In celery, the AgMAT1 transporter is discussed to be responsible for the loading of mannitol into the phloem (Noiraud et al., 2001). The same function was also suggested for the sorbitol transporters PmPLT1 and PmPLT2 from common plantain, and in fact these proteins were immunolocalized to phloem companion cells (Ramsperger-Gleixner et al., 2004). By contrast, both sorbitol transporters identified in sour cherry (PcSOT1 and PcSOT2) seem to be responsible for sorbitol import into cherries during the later stages of fruit development. Functional analyses of the encoded proteins from all three species in yeast (Noiraud et al., 2001; Gao et al., 2003; Ramsperger-Gleixner et al., 2004) and of the Plantago transporter after expression in Xenopus oocytes (Ramsperger-Gleixner et al., 2004) showed that irrespective of their physiological substrate, these proteins do catalyze the transport of both mannitol and sorbitol with similar rates.
Arabidopsis thaliana, a member of the Brassicaceae, translocates sucrose in its phloem together with small amounts of raffinose (Haritatos et al., 2000 Here, we report the isolation of cDNAs for five of these six Arabidopsis polyol transporter-like genes and the detailed characterization of one of the encoded proteins by functional expression of its cDNA in yeast and in Xenopus laevis oocytes. Our data show that in contrast with the previously described polyol transporters from polyol translocating plants, the Arabidopsis homolog AtPLT5 (At3g18830) has a strong preference for sorbitol over mannitol. However, competition analyses revealed that sorbitol transport into AtPLT5-expressing Saccharomyces cerevisiae cells was inhibited by a wide range of other compounds, including polyols with shorter chain lengths, such as xylitol, erythritol, or glycerol, the cyclic polyol myo-inositol or hexoses and pentoses forming pyranose (e.g., glucose and xylose) or furanose rings (e.g., fructose or ribose). Uptake analyses with several of these compounds in AtPLT5-expressing yeast cells and electrophysiological analyses in AtPLT5 cRNA-injected Xenopus oocytes revealed that all tested competitors are also substrates of AtPLT5. The Km values of AtPLT5 were determined for several substrates and were found to be in the millimolar range, characterizing AtPLT5 as a low affinity H+-symporter. The organ-specific expression and the cellular and subcellular localization were determined by RT-PCR analyses in AtPLT5 promoter:ß-glucuronidase (GUS) or AtPLT5 promoter:green fluorescent protein (GFP) plants and with anti-AtPLT5 antisera.
Cloning of the AtPLT cDNAs The open reading frames (ORFs) of the six putative polyol transporter genes from Arabidopsis had been predicted from in silico analyses of the Arabidopsis genome. To prove or disprove these predictions, cDNAs covering the entire ORFs were generated by RT-PCR from whole-plant mRNA and sequenced. Because of their homology to the previously described PmPLT genes from Plantago (Ramsperger-Gleixner et al., 2004 ), the Arabidopsis genes were named AtPLTs (At2g16120 [AtPLT1], At2g16130 [AtPLT2], At2g18480 [AtPLT3], At2g20780 [AtPLT4], At3g18830 [AtPLT5], and At4g36670 [AtPLT6]). The obtained cDNA sequences confirmed the predicted and deduced protein sequences of AtPLT1 (511 amino acids), AtPLT2 (511 amino acids), AtPLT3 (508 amino acids), and AtPLT5 (539 amino acids). For AtPLT4, two different ORFs had been predicted, one with 526 amino acids (e.g., NM 127643) and one with 547 amino acids (e.g., AC006234). Our analyses clearly confirmed the shorter ORF encoding the 526–amino acid AtPLT4 protein.
We were not able to isolate a cDNA for AtPLT6. The corresponding gene is predicted to encode the shortest AtPLT protein with only 493 amino acids (e.g., NM 119831). The predicted AtPLT6 intron after 100 bp of the AtPLT6 ORF was confirmed by the sequence of a full-length cDNA obtained during the large-scale cDNA sequencing of clones isolated from a hormone-treated callus (http://www.genoscope.cns.fr; BX827774). However, this clone differs strongly from the genomic AtPLT6 sequence in a downstream region of
Figure 1 presents a phylogenetic tree based on the experimentally confirmed protein sequences of AtPLT1 to AtPLT5 and on the predicted sequence of AtPLT6 (NM119831). Clearly, AtPLT1 and AtPLT2, which are encoded by adjacent genes on chromosome 2, show the highest degree of sequence conservation (93.5% identity), suggesting that one of these genes formed during a recent duplication event. AtPLT4, the most distant member of the family, shares only
Hydropathy analyses of the protein sequences predicted 12 transmembrane helices for all AtPLT proteins (data not shown) and characterized them as a separate group within the Arabidopsis monosaccharide transporter-like (MST-like) superfamily (http://www.arabidopsis.org/info/genefamily/genefamily.html/). The different lengths of the AtPLT proteins result mainly from differences in the N and C termini, with AtPLT5 (539 amino acids) having the longest C terminus, AtPLT4 (526 amino acids) having the longest N terminus, and AtPLT6 (493 amino acids) having both, the shortest N terminus and the shortest C terminus. The identity values determined for AtPLTs and polyol transporters from other species (AgMAT1, PmPLT1, or PcSOT1) are quite similar (60 to 75%), indicating the high degree of sequence conservation between these proteins from different plant species.
Expression of the AtPLT5 cDNA in S. cerevisiae
Figure 2 shows that YKY1 cells imported 14C-labeled sorbitol at a high rate, whereas no uptake of sorbitol was seen in YKY4 antisense cells. However, in contrast with all previously described polyol transporters, the AtPLT5 protein did not seem to transport 14C-labeled mannitol (Figure 2). As for other H+-symporters (Sauer et al., 1990
The obvious interpretation for this observed inhibition was that glucose may also be a substrate of AtPLT5. To test this hypothesis, we transformed the yeast strain EBY.VW-4000 with the plasmids pYK23 and pYK24, yielding strains YKY5 (sense AtPLT5) and YKY8 (antisense AtPLT5). Because of multiple gene disruptions, EBY.VW-4000 has no endogenous plasma membrane transporters for D-glucose (Wieczorke et al., 1999 ) and can therefore be used to analyze the glucose transport capacity of recombinant transporters. Figure 3 shows that AtPLT5 catalyzes the uptake of 14C-labeled glucose in YKY5, whereas no transport of 14C-glucose is seen in the antisense strain YKY8, confirming that both sorbitol and glucose are substrates of AtPLT5. This capacity to transport sugars has not been described for any of the previously published plant polyol transporters.
For further analyses of the substrate specificity, we studied the uptake of 14C-sorbitol in YKY5 cells in the presence (100-fold excess) or absence of other potential substrates. We tested the inhibitory effect of the disaccharide sucrose, of hexoses and pentoses, of linear polyols with different chain lengths (one to six carbon atoms), and of the cyclic polyol myo-inositol.
Table 1 shows that uptake of 14C-sorbitol was significantly inhibited by many of the tested competitors, suggesting that AtPLT5 may represent a transporter with an unusually wide spectrum of substrates. Unlabeled sorbitol (six carbons), but also linear polyols with five (xylitol), four (erythritol), or three carbons (glycerol) strongly reduced the import of 14C-labeled sorbitol, indicating that AtPLT5 accepts a wide range of linear polyols. Inhibition by a 100-fold excess of mannitol was less pronounced (only
Unexpectedly strong inhibition of 14C-sorbitol uptake was observed also in the presence of a 100-fold excess of myo-inositol, of different pentoses, such as xylose and arabinose, and even of ribose, which forms a furanose ring. No significant inhibition was obtained in the presence of the disaccharide sucrose.
Table 1 also shows the sensitivities of AtPLT5-dependent transport to uncouplers, such as carbonyl cyanide-m-chlorophenylhydrazone and dinitrophenol, and the effect of the SH-group inhibitor p-(chloromercuri)benzene sulfonic acid (PCMBS). Clearly, both uncouplers strongly reduced the transport rates, suggesting that AtPLT5 may catalyze the energy-dependent H+-symport of sorbitol across the yeast plasma membrane. By contrast, PCMBS had no inhibitory effect on AtPLT5-dependent transport (Table 1). This agrees with the results published for most of the polyol transporters from polyol translocating plants (Noiraud et al., 2001
To confirm that the reduced transport rates in the presence of other polyols or sugars (Table 1) result indeed from competition for transport, we analyzed AtPLT5-dependent uptake for selected radiolabeled competitors. In fact, all of the tested monosaccharides (two hexoses and two pentoses) turned out to be transported by AtPLT5 at similar rates (Figure 3). This was an important observation because so far significant transport rates for ribose have not been shown for any of the high-affinity, STP-like monosaccharide transporters from Arabidopsis (Sauer et al., 1990 Figure 3 shows AtPLT5-driven uptake also for glycerol and even for mannitol when analyzed in YKY5 cells. Together with the competition analyses in Table 1, this demonstrates that AtPLT5 can transport linear polyols with chain lengths of three to six carbons. So far, energy-dependent uptake mechanisms for glycerol have not been described for plant cells. The low rates of mannitol transport correlate with its small inhibitory effect on 14C-sorbitol uptake (Table 1), and with the almost undetectable mannitol transport rates seen in Figure 2. Obviously, AtPLT5 is expressed to higher levels in YKY5 cells than in YKY1 cells (see also the difference in sorbitol uptake in Figures 2 [YKY1] and 3 [YKY5]). This difference is attributable to the two yeast strains used for these expression analyses (SEY2102 and EBY.VW-4000). Unexpectedly, even 14C-inositol was transported with higher rates by YKY5 cells than by YKY8 antisense cells (Figure 3). However, a rather high inherent transport activity for this substrate was also seen in YKY8 control cells.
The Km values of recombinant AtPLT5 in yeast were determined for sorbitol (0.5 ± 0.1) and glucose (1.5 ± 0.8) (Figure 4). These values (mean of three analyses) agree with slightly higher sorbitol transport for sorbitol than for glucose (Figure 3). These data also show that the Km value for glucose of AtPLT5 is clearly higher than the Km values previously determined for the members of the high-affinity AtSTP monosaccharide transporters having Km values for glucose between 10 and 150 µM (Sauer et al., 1990
Expression of the AtPLT5 cDNA in X. laevis Oocytes The fact that sorbitol can be accumulated by AtPLT5-expressing yeast cells to intracellular concentrations exceeding the initial outside concentrations (dashed line in Figure 2) as well as the sensitivity of AtPLT5-driven polyol transport to uncouplers (Table 1) provides indirect evidence for an energy-dependent uptake mechanism. For a direct analysis of its energy dependence and for further analyses of its substrate specificity, AtPLT5 was analyzed in Xenopus oocytes injected with AtPLT5 cRNA (Figure 5).
Figure 5A shows that similar inward currents were obtained upon perfusion with 3-mM solutions of sorbitol, glucose, fructose, and myo-inositol at an extracellular pH of 5.5. Smaller currents were observed in the presence of 3-mM glycerol and currents almost zero in the presence of mannitol (Figure 5A). This and the normalized values shown in Figure 5B confirm the yeast data for sorbitol and glucose transport (Figure 3) and demonstrate that fructose and inositol also are transported at similar rates. By contrast, and in agreement with the data obtained in the yeast expession system (Figure 3), glycerol is transported at a clearly lower rate (  20%). These data confirm that AtPLT5 is also a transporter for myo-inositol and glycerol, two substrates that inhibited the uptake of 14C-sorbitol in yeast (Table 1) but yielded only low transport rates (glycerol in Figure 3) or were difficult to analyze because of a strong inherent transport activity (myo-inositol in Figure 3). The results also confirm that in contrast with all previously described polyol transporters, AtPLT5 does discriminate between sorbitol and mannitol. Finally, the obtained inward currents demonstrate that a positive charge is symported with each of the tested substrates, confirming the interpretation that AtPLT5-driven transport is energy dependent.
Figures 5C and 5D show the Michaelis-Menten kinetics of AtPLT5 for myo-inositol and glycerol in Xenopus oocytes. The Km values were determined at –60 mV and at an extracellular pH of 5.5 and were 3.5 ± 0.3 mM for myo-inositol and 23.4 ± 2.3 mM for glycerol. Additional analyses of the Km values for myo-inositol from 40 to –140 mV revealed that it is voltage ( The nature of the cotransported ion was characterized for glucose (Figures 6A and 6B) and glycerol (Figures 6C and 6D). In Na+-free buffer systems, inward currents induced by both substrates increased with decreasing extracellular pH values, indicating that the cotransported ions are protons and characterizing AtPLT5 as an H+-symporter. Figures 6B and 6D show that at extracellular pH values >7, the activity of AtPLT5 is almost zero. An identical pH dependence was obtained for sorbitol uptake in yeast, where transport rates for sorbitol at pH 5.0 and pH 7.0 differed >90% (data not shown).
Transient Expression of an AtPLT5-GFP Construct Both in the yeast system and in the Xenopus system, AtPLT5 activity was clearly localized to the plasma membrane. The subcellular localization of AtPLT5 in plant cells was analyzed by transient expression of an AtPLT5 construct that carried an in-frame GFP fusion at its 3'-end. To this end, the plasmid pYK25 that drives expression of this AtPLT5-GFP fusion under the control of an enhanced 35S promoter was used for transient expression in Arabidopsis and onion epidermis cells after transformation with a self-made particle inflow gun. Analyses of AtPLT5-GFP–expressing Arabidopsis (Figure 7A) and onion (Figure 7B) epidermis cells with a confocal microscope showed that fluorescence resulting from this fusion is confined to the plasma membrane. Thus, AtPLT5 is s plasma membrane–localized H+-symporter.
Analysis of AtPLT5 Expression in AtPLT5 Promoter:GUS and AtPLT5 Promoter:GFP Plants For analysis of the tissue specificity of AtPLT5 expression, we generated and analyzed AtPLT5 promoter:GUS and AtPLT5 promoter:GFP plants. A 1551-bp promoter fragment was used to drive the expression of GUS or GFP in plants that had been selected for BASTA resistance after transformation with the plasmids pYK10 (AtPLT5 promoter:GUS) or pYK13 (AtPLT5 promoter:GFP). We obtained numerous GUS- or GFP-expressing transformants and analyzed 24 independent AtPLT5 promoter:GUS lines and 24 independent AtPLT5 promoter:GFP lines. Figures 8A to 8C show the typical GUS staining found in rosette leaves of AtPLT5 promoter:GUS plants. In all plants analyzed and in most leaves of these plants, GUS staining was detected in or along the vascular tissue. However, typically this staining, which was frequently seen only after a prolonged period of staining, was patchy (Figures 8A and 7C) and often more or less absent from the midrib. In other leaves, however, GUS staining was observed mainly in the midrib (Figure 8B), and sometimes the leaves stayed completely white. Weak GUS staining was also seen along the vascular strands of stems (data not shown), of all sepals (Figure 8E), and of siliques (data not shown). Siliques typically showed an additional, more general staining near both ends (Figure 8D). Distinct AtPLT5 promoter activity was also seen in the axillary regions of all flowers (arrows in Figure 8E). In none of the tested lines was GUS staining seen in seeds, pollen, or petals (data not shown).
In 100% of the plants, the strongest GUS staining was detected over a distance of 300 to 500 µm behind the root tip (Figure 8F). Weaker GUS staining was also seen in the upper part of most roots (data not shown). Only this strong GUS activity in the tip regions could was detected in analyses of AtPLT5 promoter:GFP plants (Figure 8G). Leaf- or flower-specific expression has not been found in any of the GFP lines, suggesting that expression in these tissues is much weaker than in roots.
Immunohistochemical Analyses of AtPLT5 Localization
The quality of the obtained sera was tested on detergent extracts from total membranes isolated from YKY1 (sense) and YKY4 (antisense) yeast cells. Figure 9A shows a protein gel blot of these extracts after gel chromatographic separation and incubation with
Separation of the yeast total membrane fraction into a plasma membrane–enriched fraction and an endomembrane-enriched fraction (Sauer and Stolz, 2000 ) localized the majority of the label to the plasma membranes. The small amount of AtPLT5 protein detected in the endomembrane fraction is likely to result from contaminating plasma membranes in this fraction (Figure 9A). This localization was confirmed in immunohistochemical analyses of YKY1 and YKY4 cells, where signals were detected only at the cell surface of the YKY1 sense strain (Figure 9B). No signals were seen in sections of YKY4 antisense cells (Figure 9B). The antisera were also used in numerous attempts to immunolocalize AtPLT5 in sections of Arabidopsis roots and leaves. Unfortunately, the antisera gave no signals with different embedding and fixation protocols. In view of the specific and strong signals obtained in AtPLT5-expressing yeast cells, we speculate that the amount of AtPLT5 protein in planta is too low for immunohistochemical detection.
Analysis of AtPLT5 Expression by RT-PCR
Analysis of Mutant Plants Harboring a T-DNA Insertion in the AtPLT5 Gene Screening of publically available libraries identified a mutant line (Salk_050162) carrying a T-DNA insertion in the 4th exon of the AtPLT5 gene, 1966 bp after the start ATG (Figure 11). An insertion at this position results in a truncated AtPLT5 mRNA containing only 1374 bp of the AtPLT5 ORF (Figure 11). This mRNA is predicted to encode a protein of only 458 amino acids. In the wild-type AtPLT5 protein, the 81 C-terminal amino acids that are lacking in the truncated mutant protein encode transmembrane helix number 12 and a predicted cytoplasmic C terminus of 50 amino acids. The full-length mRNA was no longer present in the mutant line (Figure 11). The truncated AtPLT5 mRNA was, however, identified using primers binding in front of the T-DNA insertion. For the reasons given above, it is not likely that this mRNA encodes a functional protein.
When the phenotypes of this mutant line and of the corresponding wild-type plants were compared, we observed no differences in the growth phenotype under ambient CO2 concentrations and under the normal growth conditions described in Methods. Growth of mutant and wild-type plants was also compared under salt stress (100 mM NaCl), under drought stress on soil, and finally on Petri plates in the presence or absence of one of the transported substrates (10 and 50 mM sorbitol, inositol, or glycerol; data not shown). Under none of these conditions was a significant difference in plant growth or development observed.
This article describes the first member of a new class of plasma membrane–localized H+-symporters from Arabidopsis exhibiting a quite unusual substrate specificity. AtPLT5 represents the first transporter catalyzing the transport of substrates, such as myo-inositol, glycerol, or ribose across this membrane. AtPLT5 represents one of six transporters that form one (AtPLT1 to AtPLT6) of seven subfamilies of the MST-like superfamily in Arabidopsis, which was named after the intensively characterized AtSTP gene family that encodes 14 different plasma membrane–localized monosacharide transporters (Sauer et al., 1990 ; Büttner and Sauer, 2000).
So far, only members of the STP subfamily of the Arabidopsis MST-like superfamily were characterized by functional expression, although individual members of several other subfamilies have been studied by several groups (At1g08930 [AtERD6], a dehydration-induced gene, Kiyosue et al., 1998 Here, a detailed functional characterization of AtPLT5 (At3g18830) after expression of its cDNA in bakers yeast and in Xenopus oocytes is presented. In both expression systems, AtPLT5 was characterized as a polyol/cyclitol/monosaccharide-H+-symporter that is able to catalyze the energy-dependent membrane passage of a wide range of linear polyols (three to six carbon backbone), of cyclic polyols (myo-inositol), and of numerous monosaccharides, including pyranose ring-forming and furanose ring-forming hexoses and pentoses (Figures 3 and 5A).
AtPLT5 Is a Plasma Membrane–Localized Plant Transporter That Mediates Transport of Inositol, Ribose, or Glycerol
In animals, a Na+/myo-inositol symporter gene, SMIT1, has been cloned already more than 20 years ago (Kwon et al., 1992
Glycerol transport activity across plant plasma membranes has so far only been shown for members of the aquaporin family (Weig and Jakob, 2000
Finally, no transporter for ribose has been characterized so far in plant plasma membranes. Although numerous plant monosaccharide transporter genes and cDNAs have been cloned over the last years (Williams et al., 2000
A high-affinity transporter for ribose has previously been described in E. coli (Iida et al., 1984
AtPLT5 and the other five members of the AtPLT family share significant homology with known polyol transporters catalyzing the transport of mannitol or sorbitol in polyol translocating plants (Noiraud et al., 2001
AtPLT5 Is a H+-Symporter
AtPLT5 Is Expressed in Most Plant Tissues In our light cycler analyses (Figure 10), we analyzed AtPLT5 mRNA levels in several Arabidopsis tissues and especially the expression in distal and proximal regions of roots. As in the microarray analyses mentioned above, we found only marginal differences between the AtPLT5 expression levels in roots, leaves, and stems. The higher expression levels in the distal parts of the root confirmed the strong GUS activity in Figure 8F. In summary, our data suggest that AtPLT5 is expressed at rather low levels in most tissues. Our GUS and GFP analyses identify those parts of the plant where expression levels are increased above this basic level. The data are in agreement with microarray analyses.
What Is the Physiological Substrate of AtPLT5?
The affinities of AtPLT5 were shown to be 0.5 mM for sorbitol and 1.5 mM for glucose in the yeast system (Figure 4). The affinities for myo-inositol and for glycerol were determined in Xenopus oocytes, and at a constant A simple explanation for the function of ATPLT5 might be the retrieval of multiple substrates from the apoplast. All identified substrates are major components of the cellular metabolism and may leak out of the cells. However, more specific physiological functions (e.g., in the cell-to-cell distribution of certain compounds, possibly of different substrates in different tissues) certainly can not be excluded.
Strains and Growth Conditions Arabidopsis thaliana plants were grown in growth chambers on potting soil under a 16-h-light/8-h-dark regime at 22°C and 60% relative humidity or in the greenhouse under ambient conditions. For heterologous expression of AtPLT5 cDNAs in yeast, we used strains SEY2102 (Emr et al., 1983 ) or EBY.VW-4000 (Wieczorke et al., 1999). The Escherichia coli strain DH5 (Hanahan, 1983) was used for all cloning steps. Transformation of Arabidopsis was performed using Agrobacterium tumefaciens strain GV3101 (Holsters et al., 1980).
cDNA Cloning and Constructs for Expression in Yeast
Heterologous Expression in Xenopus laevis Oocytes
AtPLT5 Promoter:GUS and of AtPLT5 Promoter:GFP Constructs and Plant Transformation
Transient Expression of AtPLT5-GFP The AtPLT5 coding sequence was PCR amplified using the primers AtPLT5-NcoI-5 (5'-GACACACCATGGGAATGACAGGTGCCACACCGGAAAA-3') and AtPLT5-NcoI-3 (5'-GACACACCATGGAACTTTGTGTGTCTCCTT-3'). These primers introduced an NcoI site in front of the AtPLT5 start ATG and a second NcoI site after the last amino acid of the AtPLT5 ORF. This second NcoI site also replaced the stop codon of the original AtPLT5 sequence. The modified AtPLT5 ORF was inserted into the unique NcoI cloning site covering the start ATG of the GFP ORF in the pSO35e plasmid. The correct insertion was confirmed by sequencing, the resulting plasmid was named pYK25. For transient expression of AtPLT5-GFP from pYK25, 5 mg of surface-sterilized (70% ethanol [v/v]) gold particles (0.3- to 3-µm diameter) were resuspended in 50 µL of water and mixed with 10 µL of pYK25 DNA (1 µg/µL in water). To this mixture, 50 µL 2.5-M CaCl2 and 20 µL of 0.1-M spermidin were added. After the stepwise addition of 100 µL 100% ethanol (–20°C) and of 200 µL 100% ethanol (–20°C), the mixture was kept at –20°C for 30 min and centrifuged for 1 min at 14.000g, and gold particle–attached DNA was resuspended in 50 µL of water (DNA/gold solution). For bombardment, 9 µL of the DNA/gold solution were pipetted on a filter that was placed into the discharge assembly of a vacuum chamber (0.2 bar). The lower epidermis of Arabidopsis leaves or the inner epidermis of white onion peels (obtained from local markets) were high pressure-sprayed with the DNA/gold solution (using 50-ms helium pulses of 7, 8, or 9 bar). Sprayed leaves and onion peels were incubated for 24 to 48 h on agar plates with MS medium (Sigma-Aldrich, Deisenhofen, Germany) containing 0.3-M mannitol. Fluorescence was detected with a confocal microscope (Leica TCS SP II; Leica Microsystems, Bensheim, Germany).
Immunohistochemical Techniques and Protein Gel Blot Analyses
Protein extracts of different membrane fractions from bakers' yeast were prepared as described (Sauer and Stolz, 2000
RT-PCR and Real-Time RT-PCR
Epifluorescence and Confocal Microscopy
We thank Anja Schillinger and Jennifer Tebart for excellent technical assistance, Christian Lauterbach for helping with the confocal microscopy, and Angelika Wolf for growing the Arabidopsis plants. We are grateful to Andrea Feuerstein and Michael Büttner (both of Molecular Plant Physiology, FAU Erlangen-Nürnberg, Germany) for the pAF1 and pSO35e vectors. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to N.S. (Arabidopsis Functional Genomics Network; Sa 382/13-1).
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Norbert Sauer (nsauer{at}biologie.uni-erlangen.de). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.104.026641. Received August 5, 2004; accepted October 15, 2004.
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) dependent and increases with more negative potentials. At –140 mM, the Km of AtPLT5 for myo-inositol is 
