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First published online April 7, 2006; 10.1104/pp.106.077123 Plant Physiology 141:565-577 (2006) © 2006 American Society of Plant Biologists Arabidopsis INOSITOL TRANSPORTER4 Mediates High-Affinity H+ Symport of Myoinositol across the Plasma Membrane1Molekulare Pflanzenphysiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, D–91058 Erlangen, Germany (S.S., A.S., M.G., N.S.); Julius-von-Sachs-Institut für Biowissenschaften, Molekulare Pflanzenphysiologie und Biophysik, D–97082 Wuerzburg, Germany (K.R.K., R.H.); and Institut für Pflanzengenetik und Kulturpflanzenforschung, D–06466 Gatersleben, Germany (M.-R.H.)
Four genes of the Arabidopsis (Arabidopsis thaliana) monosaccharide transporter-like superfamily share significant homology with transporter genes previously identified in the common ice plant (Mesembryanthemum crystallinum), a model system for studies on salt tolerance of higher plants. These ice plant transporters had been discussed as tonoplast proteins catalyzing the inositol-dependent efflux of Na+ ions from vacuoles. The subcellular localization and the physiological role of the homologous proteins in the glycophyte Arabidopsis were unclear. Here we describe Arabidopsis INOSITOL TRANSPORTER4 (AtINT4), the first member of this subgroup of Arabidopsis monosaccharide transporter-like transporters. Functional analyses of the protein in yeast (Saccharomyces cerevisiae) and Xenopus laevis oocytes characterize this protein as a highly specific H+ symporter for myoinositol. These activities and analyses of the subcellular localization of an AtINT4 fusion protein in Arabidopsis and tobacco (Nicotiana tabacum) reveal that AtINT4 is located in the plasma membrane. AtINT4 promoter-reporter gene plants demonstrate that AtINT4 is strongly expressed in Arabidopsis pollen and phloem companion cells. The potential physiological role of AtINT4 is discussed.
In higher plants, cyclic polyol myoinositol and derivatives of this compound play important roles in several metabolic pathways and under several physiological conditions. For example, myoinositol represents a precursor in the synthesis of the nucleotide sugar UDP-GlcUA, which in turn is used for the synthesis of GalUA, Xyl, apiose, and Ara, important residues of plant cell wall polymers (Loewus and Murthy, 2000  ; Kanter et al., 2005). As an integral part of galactinol, myoinositol acts as a carrier of activated Gal that is eventually transferred to Suc, yielding raffinose and its longer derivatives (Kandler and Hopf, 1982). Myoinositol has also been discussed as an initial substrate in a newly described biosynthetic route for L-ascorbic acid synthesis (Lorence et al., 2004). Moreover, phytic acid (myoinositol-1,2,3,4,5,6-hexakisphosphate), the product of inositol phosphorylation (Shi et al., 2005), represents a storage form for both inositol and phosphorus, is involved in mineral storage, and may even represent a cellular energy currency (Raboy, 2003). Finally, as a component in glycosylphosphatidylinositol-membrane anchors (Schultz et al., 1998) and especially as a component of phospholipids (Lehle, 1990), inositol is involved not only in providing the structural basis for membranes and membrane-attached proteins, but also in several signaling pathways.
In many plants, the concentration of myoinositol is massively increased in response to salt stress. The reason is that compounds such as myoinositol, pinitol, or ononitol, can be accumulated to high concentrations as osmolytes (Thomas and Bohnert, 1993
Despite these essential functions of myoinositol in plant biology and despite the profound knowledge of myoinositol metabolism in plants, little is known about the partitioning of this molecule between different cells, tissues, or subcellular compartments. Nelson and coworkers (1999)
The first cDNAs of putative plant myoinositol transporters were cloned from ice plant. One of these cDNAs (Mesembryanthemum INOSITOL TRANSPORTER1 [MITR1]) was expressed in a mutant line of yeast (Saccharomyces cerevisiae; Chauhan et al., 2000
Only recently, a plasma membrane-localized transporter from Arabidopsis was shown to mediate the uptake of a wide range of different substrates, including monosaccharides (pentoses and hexoses), linear polyols of different chain lengths (3–6 carbons), and myoinositol (AtPLT5; Klepek et al., 2005
In this study, we concentrated on the analysis of one of four Arabidopsis proteins sharing homology with MITR1. These proteins represent an independent subgroup within the Arabidopsis MST-like superfamily. All previously characterized proteins of this superfamily were shown to be H+ symporters (AtSTP1 [Sauer et al., 1990
Here we present the cloning, functional characterization, tissue-specific expression, and subcellular localization of Arabidopsis INOSITOL TRANSPORTER4 (AtINT4). AtINT4 is one of four highly homologous proteins from Arabidopsis and transports myoinositol with high specificity. AtINT4 is localized in the plasma membrane and expression of AtINT4 cDNA in both yeast and Xenopus independently confirmed that it transports myoinositol with high affinity and high specificity. Xenopus expression data show that AtINT4 is an H+ symporter. The physiological role of the protein was studied with AtINT4 promoter-
Cloning of AtINT cDNAs
In silico analyses of the Arabidopsis genome identified four open reading frames (ORFs) for putative transporters sharing homology with MITR1 (accession no. AF280431) and MITR2 (accession no. AF280432) from ice plant (Chauhan et al., 2000 The obtained cDNA sequences confirmed the predicted protein sequences of AtINT2 (580 amino acids) and AtINT4 (582 amino acids). For AtINT1, two different ORFs had been predicted, one with 509 amino acids (accession no. NP_850393) and one with 521 amino acids (accession no. AAB64332). Our analyses confirmed the shorter ORF for an AtINT1 protein with 509 amino acids. We were not able to amplify a cDNA for AtINT3 that is predicted to encode a protein of 580 amino acids (accession no. NP_181117) that is similar to AtINT2 and AtINT4 with 60.4% and 63.4% identical amino acids. The obtained cDNA sequences for AtINT1, AtINT2, and AtINT4 were submitted to the EMBL Nucleotide Sequence Database (accession nos. AtINT1, AJ973175; AtINT2, AJ973176; and AtINT4, AJ973178). Hydropathy analyses of the deduced protein sequences predicted 12 transmembrane helices for all AtINT proteins (data not shown) and sequence comparisons with other transporters characterized them as a separate group within the Arabidopsis MST-like superfamily (http://www.arabidopsis.org/info/genefamily/genefamily.html). Figure 1 presents a phylogenetic tree based on the experimentally confirmed protein sequences of AtINT1, AtINT2, and AtINT4 and on the predicted sequence of AtINT3 (NP_181117). AtINT4 is closely related to ice plant MITR proteins. One of the AtINT proteins (AtINT1) is much smaller than all other proteins in Figure 1 (509 amino acids versus 580 to 584 amino acids). This difference results from an 80-amino acid fragment that is missing in the loop between the predicted transmembrane helices IX and X of AtINT1. Hydropathy analyses (data not shown) predict this loop to be opposite the N and C termini (i.e. outside the cell in a plasma membrane transporter or inside the vacuole in a vacuolar transporter). There are two consensus sequences for N-glycosylation in AtINT4 (Asn312-Lys313-Thr314 and Asn385-Ala386-Thr387). Consensus sequences are located between predicted transmembrane helices VII and VIII or in the large loop between predicted transmembrane helices IX and X. Thus, these Asn residues do face the lumen of the endoplasmic reticulum and may be N-glycosylated during protein biosynthesis. Both glycosylation sites are conserved in AtINT2 and AtINT3, but are missing from AtINT1, which has no consensus sequence for N-glycosylation.
The calculated pI (IP) of AtINT4 is 8.70. This value is similar to the values for AtINT2 (IP = 8.25) and AtINT3 (IP = 7.74) and different from the value for AtINT1 (IP = 5.05). The IP values of AtINT2, AtINT3, and AtINT4 also resemble the quite basic IP values of numerous plasma membrane transporters (e.g. AtSTP1 [Sauer et al., 1990 ; IP = 9.49]; AtSUC2 [Sauer and Stolz, 1994; IP = 9.55]; and AtPLT5 [Klepek et al., 2005; IP = 10.39]).
To determine the functional properties of this new group of Arabidopsis transporters, we cloned the cDNA of AtINT4, the first full-length clone obtained, into the unique EcoRI site of the yeast expression vector NEV-E-Leu, which is a modification of NEV-E (Sauer and Stolz, 1994 The resulting yeast strains SSY33 (sense AtINT4) and SSY34 (antisense AtINT4) were grown on petri plates containing low (2 µg/mL) or high (20 µg/mL) concentrations of myoinositol to study the possible complementation of the mutations in myoinositol uptake and biosynthesis. Figure 2 shows that SSY33 cells had regained the capacity to grow on low myoinositol, whereas hardly any growth was seen for control strains (SSY35 with the empty vector or SSY34 with the antisense plasmid) on the same medium. This was evidence that AtINT4 does encode a myoinositol transporter.
Chauhan et al. (2000) had used the same yeast mutant for complementation with MITR1 and got a similar result. In addition, these authors observed a growth-accelerating effect in the presence of 10 mM NaCl, and this was one of the observations that finally led to the interpretation that MITR1 is a Na+/inositol symporter. We tested whether this is also seen in AtINT4-expressing yeast cells (Fig. 2). In fact, SSY33 cells do grow significantly better on plates supplemented with 10 mM NaCl. With SSY33, we tried to determine the Km value, substrate specificity, and sensitivity of AtINT4 to uncouplers. However, transport rates determined with 3H-labeled myoinositol were too low to allow such analyses (data not shown). Apparently, the amount of AtINT4 protein in the yeast plasma membrane is sufficiently high for complementation, but not high enough to allow direct measurement of radiolabeled substrates. In fact, when an AtINT4-GFP fusion was expressed in baker's yeast, most of the GFP fluorescence remained inside the cells, most likely in the endoplasmic reticulum (data not shown). For further analyses of the AtINT4 transport properties, we had to use a different expression system.
X. laevis oocytes are a perfect tool for analyses of electrogenic plant plasma membrane transporters (Aoshima et al., 1993
Figure 3A
shows a recording of inward currents in the presence of the different substrates tested. Analyses were performed with 10 mM solutions of myoinositol, Suc, sorbitol, Glc, or Fru at an extracellular pH of 5.5. Only myoinositol gave a strong inward current. No significant currents were obtained with any of the other compounds. This confirms and extends the results obtained in the yeast complementation analysis, showing that AtINT4 does mediate import of myoinositol into injected Xenopus oocytes. Furthermore, the obtained inward currents demonstrate that a positive charge is symported with myoinositol, confirming that AtINT4-driven transport is energy dependent. Finally, this result shows that, in contrast to the previously described AtPLT5 transporter, which has low substrate specificity (monosaccharides, linear polyols, and even myoinositol), AtINT4 seems to be highly specific for myoinositol. In addition to the compounds tested in Figure 3A, no significant currents were obtained by mannitol, xylitol, Xyl, Gal, Ara, erythritol, inositol-6-P, Rib, glycerol, or gluconate (each n
The identity of the cotransported ion was determined by replacing individual ions in the extracellular solution. There was no difference between the currents elicited with 95 mM KCl/0 mM NaCl or with 95 mM NaCl/0 mM KCl (data not shown); also, the simultaneous replacement of K+ and Na+ by N-methylglucamine+ (NMG-Cl) had no effect on the resulting inward currents (Fig. 3B; n = 9). This demonstrates that protons are cotransported ions and that AtINT4 is a myoinositol/H+ symporter.
Figure 3C shows one of 10 independent measurements where the inward H+ currents elicited by myoinositol decreased with increasing extracellular pH. Similar pH dependence had been described for the AtPLT5 PLT, which has only recently been characterized as an H+ symporter (Klepek et al., 2005
A typical substrate dependence of AtINT4 for myoinositol is shown in Figure 3D and the corresponding Michaelis-Menten kinetics are presented in Figure 3E. Km values were determined at three different membrane potentials (–90 mV, –40 mV, and 0 mV) at an extracellular pH of 5.5. Unexpectedly, there was no difference at these different membrane potentials and an average Km of 0.24 ± 0.01 mM could thus be calculated from 10 independent analyses (Fig. 3E). This value is significantly lower than the Km for myoinositol determined for AtPLT5 in Xenopus oocytes (3.5 ± 0.3 mM; Klepek et al., 2005
For analysis of the tissue specificity of AtINT4 expression, we generated and analyzed AtINT4 promoter-GUS and AtINT4 promoter-GFP plants. A 1,518-bp promoter fragment was used to drive the expression of GUS or GFP in plants that had been selected for gluphosinate resistance after transformation with the plasmids pLEX110 (AtINT4 promoter GUS) or pLEX164 (AtINT4 promoter GFP). We obtained numerous GUS- or GFP-expressing transformants and analyzed 24 independent AtINT4 promoter-GUS lines and 24 independent AtINT4 promoter-GFP lines. Based on microarray analyses, strong and specific expression of AtINT4 had been predicted in Arabidopsis stamina (https://www.genevestigator.ethz.ch). Our AtINT4 promoter-GUS and AtINT4 promoter-GFP plants confirmed this prediction (Fig. 4, A, G, and H ), and more detailed analyses revealed that the strong GUS-histochemical staining and GFP fluorescence in anthers is restricted to pollen (Fig. 4, B and H). The well-known autofluorescence of Arabidopsis pollen that is detected under the same conditions (data not shown) was much weaker and had a clearly distinguishable color (yellowish).
In addition to the promoter activity in pollen, we also observed GUS staining and GFP fluorescence in the leaves of these plants (Fig. 4, C–E and I). This leaf-specific promoter activity was restricted to the vascular tissue and absent from the mesophyll (Fig. 4, D and I). It was also seen in the vascular tissues of cotyledons, hypocotyls, and roots (Fig. 4, C and E). This vascular-specific AtINT4 expression in leaves depended strongly on the developmental stage. No promoter activity was found in young sink leaves (Fig. 4, C and E, red arrows), whereas strong expression was seen in all veins of fully developed source leaves (Fig. 4C). During the sink-to-source transition (transition leaves), AtINT4 expression started in the very tips of the leaves (Fig. 4, C and E, black arrows) and proceeded toward the petioles during leaf development. This expression pattern (pollen and vascular tissue) was observed in 22 of 24 analyzed AtINT4 promoter-GUS plants and in 23 of 24 analyzed AtINT4 promoter-GFP plants. For further characterization of the precise tissue expressing AtINT4 within the vascular tissue, we analyzed cross sections of AtINT4 promoter-GUS leaves. Figure 4F shows a cross section through the midrib of a leaf from an AtINT4 promoter-GUS plant, which shows that GUS staining is restricted to the phloem.
Antisera were raised against the peptide NH2-LLEVGFKPSILRRREKKGKEVDAA-COOH that corresponds to amino acids 559 to 582 at the very C terminus of AtINT4. This sequence was not found in any other Arabidopsis protein. The quality of the obtained sera was tested on detergent extracts from total membranes isolated from yeast strains ScLEX30 (harbors plasmid NEV-N [Sauer and Stolz, 2000
The difference between the apparent molecular mass of the monomeric form (55 kD) and the molecular mass deduced from the DNA sequence (62.9 kD) was unexpected. Typically, lipophilic proteins run at higher apparent molecular masses on SDS gels, which results from excess binding of SDS (Beyreuther et al., 1980 ; Gahrtz et al., 1994; Barth et al., 2003). A reduced apparent molecular mass may result from protein modification (e.g. by proteolytic cleavage).
In a next step, the In contrast to the GUS and GFP data (Fig. 4, B and H), no immunosignals were obtained in mature pollen grains that were still in the anthers. This suggests that the AtINT4 promoter is active in these pollen grains to form AtINT4 mRNA, but that this mRNA is translated only after hydration of the pollen on Arabidopsis stylar tissue.
The subcellular localization of AtINT4 in planta was analyzed using an AtINT4 protein with GFP fused to its C terminus. To this end, the plasmid pSS24 that drives expression of the AtINT4-GFP fusion under the control of an enhanced 35S promoter was used for transient expression in Arabidopsis protoplasts (Fig. 6, A and B ) or in particle-bombarded epidermis cells of tobacco (Fig. 6C). AtINT4-GFP-expressing cells and protoplasts were analyzed with a confocal microscope. In all analyses, the red autofluorescence of the chloroplasts was located inside the GFP-labeled structure (Fig. 6, A–C). No GFP fluorescence was found in any other structure inside the transformed cells, indicating that, in both plant expression systems (Arabidopsis and tobacco), the AtINT4-GFP fusion protein is located in the plasma membrane.
Analysis of Mutant Plants Harboring a T-DNA Insertion in the AtINT4 Gene Screening of publicly available libraries identified a mutant line (Salk_082661, Atint4-2) with a T-DNA insertion in the second exon of the AtINT4 gene, 929 bp after the start ATG (Fig. 7A ). We performed PCR reactions to identify homozygous Atint4-2 plants (e.g. plant 2 in the PCR shown in Fig. 7A) and used these plants for further analyses. Homozygous mutant plants showed a complete loss of intact AtINT4 mRNA (Fig. 7B, i), whereas partial AtINT4 mRNAs resulting from the sequences flanking the T-DNA insertion could be amplified from wild-type and Atint4-2 RNA preparations. The 840-bp fragment flanking the left border of the T-DNA insertion (Fig. 7B, 5' end) is likely to result from AtINT4-promoter transcriptional activity. The 314-bp fragment flanking the predicted (but not sequenced) right border of the T-DNA insertion (Fig. 7B, 3' end) most likely results from a promoter activity within the T-DNA insertion. Nevertheless, growth analyses under standard conditions (on soil in the growth chamber: 21°C, 60% relative humidity, long day [16-h light/8-h dark] or short day [8-h light/16-h dark]), on different concentrations of NaCl (10–100 mM), or on different concentrations of myoinositol in the growth medium (0–100 mM) revealed no phenotypic differences between the T-DNA insertion line and the isogenic wild type. Also, the fertility of the plants was not affected by the T-DNA insertion in AtINT4.
This article presents a detailed characterization of AtINT4 (At4g16480), a high-affinity, plasma membrane-localized H+ symporter that is specific for myoinositol. AtINT4 represents one of four transporters (AtINT1–AtINT4) that form an independent subfamily within the MST-like superfamily in Arabidopsis that was named after the AtSTP gene family of plasma membrane-localized MSTs (Sauer et al., 1990 ; Büttner and Sauer, 2000).
So far, only members of the sugar transporter and the PLT subfamilies of the Arabidopsis MST-like superfamily were characterized by functional expression. Individual members of several other subfamilies have been studied (At1g08930 [AtERD6], an early dehydration induced gene [Kiyosue et al., 1998
The functional characterization of AtINT4 is based on expression of its cDNA in bakers' yeast and Xenopus oocytes. In both systems, AtINT4 was characterized as a myoinositol transporter, in yeast by the complementation of a growth defect and in Xenopus by the analysis of currents elicited by this substrate. The latter analyses demonstrated that myoinositol is transported across the plasma membrane via an H+-symport mechanism (Fig. 3).
These analyses suggested, and the localization of AtINT4-GFP in plasma membranes of Arabidopsis and tobacco fusion confirmed (Fig. 6), that the AtINT4 protein is a plasma membrane transporter. An H+-dependent transport of myoinositol, the only identified substrate of AtINT4, has also been described for AtPLT5, another plasma membrane transporter of Arabidopsis (Klepek et al., 2005
The only plant myoinositol transporters cloned so far were identified in the common ice plant (MITR1 and MITR2; Chauhan et al., 2000
In contrast, a myoinositol transporter from the protozoan Leishmania donovani (LdD1) and a human myoinositol transporter (HMIT) were both characterized as H+ symporters by expression in Xenopus oocytes (Drew et al., 1995
In addition to the H+ symporter HMIT (Uldry et al., 2001
The report that MITR1 and MITR2 act as Na+ symporters (Chauhan et al., 2000
Using antisera that had been raised against peptides from the central cytoplasmic loop, MITR1 and MITR2 were detected in vacuolar membrane fractions (Chauhan et al., 2000
AtINT4 expression was observed in pollen grains and in the vascular tissue of AtINT4 promoter-GUS and AtINT4 promoter-GFP plants (Fig. 4). The expression of AtINT4 in pollen, but not in the vasculature, had been predicted from microarray analyses (https://www.genevestigator.ethz.ch). From Figure 4, D and I, it is obvious that gene expression in the vasculature of Arabidopsis leaves is limited to very few cells in a leaf. Moreover, analyses of AtINT4 promoter-GFP plants (Fig. 4, H and I) suggest that the relative expression of AtINT4 is stronger in pollen than in the vasculature. Together, these observations explain why vascular expression has not been seen in microarray analyses.
Attempts to confirm these expression data by immunohistochemical analyses were successful for pollen tubes. Sections through Arabidopsis stylar tissue showed FITC-dependent fluorescence only after incubation with an
Similar observations (i.e. translation of a preformed mRNA only after hydration and germination of pollen on the stylar tissue) have also been made for other genes (Linskens et al., 1970
With several sets of PCR primers it was not possible to identify a complete AtINT4 mRNA in Atint4-2 plants (Fig. 7; data not shown). Nevertheless, these plants do form fertile seeds and all other parameters analyzed (standard growth conditions, salinity stress, and varying inositol concentrations) did not differ from isogenic wild-type plants. Identification of AtINT4 mRNA fragments that correspond to AtINT4 genomic sequences flanking both sides of the T-DNA insertion raises the question of whether the encoded, truncated transporter fragments can form a functional unit. In principle, this cannot be excluded because it has been shown for other transporters that truncated proteins encoded by split mRNAs can form a functional complex (Wrubel et al., 1990
It has been reported that Arabidopsis transports small amounts of raffinose in its phloem (Haritatos et al., 2000
Analyses of AtGolS1 promoter-GUS plants (Panikulangara et al., 2004 In ice plants, myoinositol transport is thought to play a role in salt tolerance. In Arabidopsis, there is no indication for a regulation of AtINT4 expression by salt treatment (https://www.genevestigator.ethz.ch) nor is myoinositol accumulating in salt-grown Arabidopsis plants. Thus, AtINT4 does not seem to play a role in the response of Arabidopsis plants to salt treatment. Similarly, changes in the concentrations of exogenously supplied myoinositol (0 mM, 1 mM, 10 mM, and 100 mM) did not result in phenotypic differences between wild-type and Atint4-2 plants. As for many other transporter genes, the roles of specific transport activity in pollen, pollen tubes, and phloem is still unclear. For a final answer to this question, we will need double, triple, or quadruple knockout lines that are not yet available at this time.
Strains and Growth Conditions
Arabidopsis (Arabidopsis thaliana) plants were grown in a growth chamber on potting soil under a 16-h light/8-h dark regime at 22°C and 60% relative humidity or in a greenhouse under ambient conditions. For heterologous expression of AtINT4 cDNAs in yeast (Saccharomyces cerevisiae), we used strain D458-1B (Nikawa et al., 1991
cDNAs of three of four Arabidopsis AtINT genes were amplified from total RNA isolated from Arabidopsis ecotype Columbia with gene-specific primers binding to the very 5' ends (including the start ATG) or the very 3' ends (including the stop codon) of the cDNAs. NotI cloning sites were introduced at both ends of AtINT1 and AtINT2, and EcoRI cloning sites were introduced at both ends of AtINT4. The resulting cDNAs were digested with the respective enzymes, cloned into the yeast expression vectors NEV-E or NEV-N (Sauer and Stolz, 1994
For functional analysis in Xenopus laevis oocytes, the AtINT4 insert of pLEX114s was excised with EcoRI and cloned into the plasmid pDK148 (Jespersen et al., 2002
A 1,518-bp promoter AtINT4 promoter fragment was PCR amplified from genomic DNA (Arabidopsis ecotype Columbia) using the primers INT4-p5 (5'-GTCCGAAAAAGCTTGGGTTCAAATCCCACTCTTGACA-3') and INT4-p3 (5'-TCCTTCCACCATGGTTTCTTCTTGTCTGATCTCTC-3'). The primers introduced an N-terminal HindIII and a C-terminal NcoI site that were used to clone the resulting fragment in front of the ORF of GFP and a transcriptional terminator in a pUC19-based plasmid (pEPS1/pUC19; Imlau et al., 1999
For transient expression of AtINT4-GFP fusions, we used the plasmid pSO35e (Klepek et al., 2005
pSS24 was used for transient expression of AtINT4 in Arabidopsis protoplasts (polyethylene glycol transformation; modified after Abel and Theologis [1994]
The T-DNA insertion line Atint4-2 (Salk_082661) was identified using the Salk Institute T-DNA express gene-mapping tool (Alonso et al., 2003 The primers int4-5' (5'-ATATCTCTGAATTCAACAATGGTGGAAGGAGGAATTGCG-3'), int4-3' (5'-ATATCTCTGAATTCTTAAGCAGCATCGACTTCTTTGCCC-3'), int4-840r (5'-AGCAGCGAGTCCACGTCGAAC-3'), and int4-1435 (5'-CCGTGGATCGTCAACTCT-3') were used for reverse transcription-PCRs with total RNA from wild-type and mutant Arabidopsis leaves. The primers AtACT5 (5'-GCGATGAAGCTCAATCCAAACGAG-3') and AtACT3 (5'-GGTCACGACCAGCAAGATCAAGAC-3') were used to amplify the AtACT1 mRNA.
For production of anti-AtINT4 antisera (
Binding of anti-AtINT4 (
Protein extracts of total membrane fractions from bakers' yeast were prepared as described (Sauer and Stolz, 2000
To measure the concentrations of sugars and sugar alcohols, a BioLC DX600 system (Dionex) was used, including a gradient pump (GP50), a degasser module, an autosampler (AS50), and a pulsed amperometric detector (EG50). Separation of the anionic compounds was carried out using a CarboPack MA1 column (4 x 250 mm) connected to a guard column of the same material (4 x 10 mm) and an ATC-1 anion trap column, which was placed between the eluent and separation columns to remove the anionic contaminants present in the eluents. A linear gradient was accomplished with purest water (buffer A; Millipore) and 480 mM sodium hydroxide (Baker; 50% solution, buffer B). The column was equilibrated at a flow rate of 0.4 mL/min. The duration of the run was 65 min. The calibration and quantitative calculation of inositol and raffinose were carried out using chromeleon software 6.6.
Images of GFP fluorescence were made with an epifluorescence microscope (Zeiss Axioskop; Carl Zeiss GmbH), with stereomicroscopes (Zeiss SV11 or Leica MZFLIII; Leica Microsystems) with an excitation wavelength of 460 to 500 nm or with a confocal laser-scanning microscope (Leica TCS SPII). Confocal images were processed using Leica confocal software 2.5. Emitted fluorescence was monitored at detection wavelengths longer than 510 nm.
We thank Ruth Stadler for experimental help and Angelika Wolf for growing the Arabidopsis plants. Received January 13, 2006; returned for revision March 31, 2006; accepted March 31, 2006.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant 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.plantphysiol.org) is: Norbert Sauer (nsauer{at}biologie.uni-erlangen.de). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.077123. * Corresponding author; e-mail nsauer{at}biologie.uni-erlangen.de; fax 49–9131–8528751.
Abel S, Theologis A (1994) Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression. Plant J 5: 421–427[CrossRef][Web of Science][Medline] Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657 Aoshima H, Yamada M, Sauer N, Komor E, Schobert C (1993) Heterologous expression of the H+/hexose cotransporter from Chlorella in Xenopus oocytes and its characterization with respect to sugar specificity, pH and membrane potential. J Plant Physiol 141: 293–297[Web of Science] Barth I, Meyer S, Sauer N (2003) PmSUC3: characterization of a SUT2/SUC3-type sucrose transporter from Plantago major. Plant Cell 15: 1375–1385 Becker D, Dreyer I, Hoth S, Reid JD, Busch H, Lehnen M, Palme K, Hedrich R (1996) Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K+ channel KAT1. Proc Natl Acad Sci USA 93: 8123–8128 Becker D, Kemper E, Schell J, Masterson R (1992) New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol Biol 20: 1195–1197[CrossRef][Web of Science][Medline] Beyreuther K, Bieseler B, Ehring R, Griesser H-W, Mieschendahl M, Müller-Hill B, Triesch I (1980) Investigation of structure and function of lactose permease of Escherichia coli. Biochem Soc Trans 8: 675–676[Medline] Boorer KJ, Loo DD, Frommer WB, Wright EM (1996) Transport mechanism of the cloned potato H+/sucrose cotransporter StSUT1. J Biol Chem 271: 25139–25144 Borstlap AC (2002) Early diversification of plant aquaporins. Trends Plant Sci 7: 529–530[CrossRef][Web of Science][Medline] Büttner M, Sauer N (2000) Monosaccharide transporters in plants: structure, function and physiology. Biochim Biophys Acta 1465: 263–274[Medline] Capkova V, Hrabetova E, Tupy J (1988) Protein synthesis in pollen tubes: preferential formation of new species independent of transcription. Sex Plant Reprod 1: 150–155 Chauhan S, Forsthoefel N, Ran Y, Quigley F, Nelson DE, Bohnert HJ (2000) Na+/myoinositol symporters and Na+/H+-antiport in Mesembryanthemum crystallinum. Plant J 24: 511–522[CrossRef][Web of Science][Medline] Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743[CrossRef][Web of Science][Medline] Drew ME, Langford CK, Klamo EM, Russell DG, Kavanaugh MP, Landfear SM (1995) Functional expression of a myoinositol/H+ symporter from Leishmania donovani. Mol Cell Biol 15: 5508–5515[Abstract] Dunn SD (1986) Effects of the modification of transfer buffer composition on the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal Biochem 157: 144–153[CrossRef][Web of Science][Medline] Emr SD, Scheckman R, Flessel MC, Thorner J (1983) An MF Gahrtz M, Stolz J, Sauer N (1994) A phloem specific sucrose-H+ symporter from Plantago major L. supports the model of apoplastic phloem loading. Plant J 6: 697–706[CrossRef][Web of Science][Medline] Gao Z, Maurousset L, Lemoine R, Yoo SD, Van Nocker S, Loescher W (2003) Cloning, expression, and characterization of sorbitol transporters from developing sour cherry fruit and leaf sink tissues. Plant Physiol 131: 1566–1575 Gietz D, Jean WS, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20: 1425 Hanahan D (1983) Studies on transformation of E. coli with plasmids. J Mol Biol 166: 557–580[Web of Science][Medline] Haritatos E, Medville RA, Turgeon R (2000) Minor vein structure and sugar transport in Arabidopsis thaliana. Planta 211: 105–111[CrossRef][Web of Science][Medline] Hediger MA, Ikeda T, Coady M, Gundersen CB, Wright EM (1987) Expression of size-selected mRNA encoding the intestinal Na+/glucose cotransporter in Xenopus laevis oocytes. Proc Natl Acad Sci USA 84: 2634–2637 Holsters M, Silva B, Van Vliet F, Genetello C, De Block M, Dhaese P, Depicker A, Inze D, Engler G, Villarroel R, et al (1980) The functional organization of the nopaline Agrobacterium tumefaciens plasmid pTiC58. Plasmid 3: 212–230[CrossRef][Web of Science][Medline] Imlau A, Truernit E, Sauer N (1999) Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues. Plant Cell 11: 309–322 Jespersen T, Grunnet M, Angelo K, Klaerke DA, Olesen SP (2002) Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes. Biotechniques 32: 536–540[Web of Science][Medline] Kandler O, Hopf H (1982) Oligosaccharides based on Suc (sucrosyl oligosaccharides). In FA Loewus, W Tanner, eds, Plant Carbohydrates 1. Encyclopedia of Plant Physiology: Plant Carbohydrates I, Intracellular Carbohydrates, New Series, Vol 13 A. Springer-Verlag, Berlin, pp 348–383 Kanter U, Usadel B, Guerineau F, Li Y, Pauly M, Tenhaken R (2005) The inositol oxygenase gene family of Arabidopsis is involved in the biosynthesis of nucleotide sugar precursors for cell-wall matrix polysaccharides. Planta 221: 243–254[CrossRef][Web of Science][Medline] Kiyosue T, Abe H, Yamaguchi-Shinozaki K, Shinozaki K (1998) ERD6, a cDNA clone for an early dehydration-induced gene of Arabidopsis, encodes a putative sugar transporter. Biochim Biophys Acta 1370: 187–191[Medline] Kjellbom P, Larsson C, Johansson II, Karlsson M, Johanson U (1999) Aquaporins and water homeostasis in plants. Trends Plant Sci 4: 308–314[CrossRef][Web of Science][Medline] Klepek YS, Geiger D, Stadler R, Klebl F, Landouar-Arsivaud L, Lemoine R, Hedrich R, Sauer N (2005) Arabidopsis POLYOL TRANSPORTER5, a new member of the monosaccharide transporter-like superfamily, mediates H+-symport of numerous substrates, including myo-inositol, glycerol, and ribose. Plant Cell 17: 204–218 Kwon HM, Yamauchi A, Uchida S, Preston AS, Garcia-Perez A, Burg MB, Handler JS (1992) Cloning of the cDNA for a Na+/myo-inositol cotransporter, a hypertonicity stress protein. J Biol Chem 267: 6297–6301 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685[CrossRef][Medline] Lehle L (1990) Phosphatidyl inositol metabolism and its role in signal transduction in growing plants. Plant Mol Biol 15: 647–658[CrossRef][Web of Science][Medline] Lin J-J, Dickinson DB, Ho T-HD (1987) Phytic acid metabolism in lily (Lilium longiflorum Thunb.) pollen. Plant Physiol 83: 408–413 Linskens HF, Schrauwen JAM, Konings RNK (1970) Cell-free protein synthesis with polysomes from germinating Petunia pollen grains. Planta 90: 153–162[CrossRef] Loewus FA, Murthy PPN (2000) myo-Inositol metabolism in plants. Plant Sci 150: 1–19 Lorence A, Chevone BI, Mendes P, Nessler CL (2004) myo-Inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol 134: 1200–1205 Marger MD, Saier MH Jr (1993) A major superfamily of transmembrane facilitators that catalyze uniport, symport and antiport. Trends Biochem Sci 18: 13–20[CrossRef][Web of Science][Medline] Mascarenhas JP (1990) Gene activity during pollen development. Annu Rev Plant Physiol Plant Mol Biol 41: 317–338[CrossRef][Web of Science] Matskevitch J, Wagner CA, Risler T, Kwon HM, Handler JS, Waldegger S, Busch AE, Lang F (1998) Effect of extracellular pH on the myo-inositol transporter SMIT expressed in Xenopus oocytes. Pflugers Arch 436: 854–857[CrossRef][Web of Science][Medline] Nelson DE, Koukoumanos M, Bohnert HJ (1999) Myo-inositol-dependent sodium uptake in ice plant. Plant Physiol 119: 165–172 Nelson DE, Rammesmayer G, Bohnert HJ (1998) Regulation of cell-specific inositol metabolism and transport in plant salinity tolerance. Plant Cell 10: 753–764 Nikawa J, Tskugoshi Y, Yamashita S (1991) Isolation and characterization of two distinct myo-inositol transporter genes of Saccharomyces cerevisiae. J Biol Chem 266: 11184–11191 Noiraud N, Maurousset L, Lemoine R (2001) Identification of a mannitol transporter, AgMaT1, in celery phloem. Plant Cell 13: 695–705 Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12: 357–358 Pajor AM, Wright EM (1992) Cloning and functional expression of a mammalian Na+/nucleoside cotransporter: a member of the SGLT family. J Biol Chem 267: 3557–3560 Panikulangara TJ, Eggers-Schumacher G, Wunderlich M, Stransky H, Schöffl F (2004) Galactinol synthase1: a novel heat shock factor target gene responsible for heat-induced synthesis of raffinose family oligosaccharides in Arabidopsis. Plant Physiol 136: 3148–3158 Quirino BF, Reiter WD, Amasino RD (2001) One of two tandem Arabidopsis genes homologous to monosaccharide transporters is senescence-associated. Plant Mol Biol 46: 447–457[CrossRef][Web of Science][Medline] Raboy V (2003) myo-Inositol-1,2,3,4,5,6-hexakisphosphate. Phytochemistry 64: 1033–1043[CrossRef][Web of Science][Medline] Ramsperger-Gleixner M, Geiger D, Hedrich R, Sauer N (2004) Differential expression of sucrose transporter and polyol transporter genes during maturation of common plantain companion cells. Plant Physiol 134: 147–160 Reinders A, Panshyshyn JA, Ward JM (2005) Analysis of transport activity of Arabidopsis sugar alcohol permease homolog AtPLT5. J Biol Chem 280: 1594–1602 Reinders A, Schulze W, Thaminy S, Stagljar I, Frommer WB, Ward JM (2002) Intra- and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split-ubiquitin system and functional assays. Structure 10: 763–772[Medline] Robinson KS, Lai K, Cannon TA, McGraw P (1996) Inositol transport in Saccharomyces cerevisiae is regulated by transcriptional and degradative endocytic mechanisms during the growth cycle that are distinct from inositol-induced regulation. Mol Biol Cell 7: 81–89[Abstract] Sauer N, Friedländer K, Gräml-Wicke U (1990) Primary structure, genomic organization and heterologous expression of a glucose transporter from Arabidopsis thaliana. EMBO J 9: 3045–3050[Web of Science][Medline] Sauer N, Stolz J (1994) SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in bakers' yeast and identification of the histidine tagged protein. Plant J 6: 67–77[CrossRef][Web of Science][Medline] Sauer N, Stolz J (2000) Expression of foreign transport proteins in yeast. In SA Baldwin, ed, Practical Approach Series. Oxford University Press, New York, pp 79–105 Schultz C, Gilson P, Oxley D, Youl J, Bacic A (1998) GPI-anchors on arabinogalactan-proteins: implications for signalling in plants. Trends Plant Sci 3: 426–431[CrossRef][Web of Science] Sheveleva E, Chmara W, Bohnert HJ, Jensen RG (1997) Increased salt and drought tolerance by D-ononitol production in transgenic Nicotiana tabacum. Plant Physiol 115: 1211–1219[Abstract] Shi J, Wang H, Hazebroek J, Ertl DS, Harp T (2005) The maize low-phytic acid 3 encodes a myo-inositol kinase that plays a role in phytic acid biosynthesis in developing seeds. Plant J 42: 708–719[CrossRef][Web of Science][Medline] Stadler R, Truernit E, Gahrtz M, Sauer N (1999) The AtSUC1 sucrose carrier may represent the osmotic driving force for anther dehiscence and pollen tube growth in Arabidopsis. Plant J 19: 269–278[CrossRef][Web of Science][Medline] Taji T, Ohsumi C, Iuchi S, Seki M, Kasuga M, Kobayashi M, Yamaguchi-Shinozaki K, Shinozaki K (2002) Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. Plant J 29: 417–426[CrossRef][Web of Science][Medline] Thomas JC, Bohnert HJ (1993) Salt stress perception and plant growth regulators in the halophyte Mesembryanthemum crystallinum. Plant Physiol 103: 1299–1304[Abstract] Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24: 4876–4882 Truernit E, Sauer N (1995) The promoter of the Arabidopsis thaliana SUC2 sucrose-H+ symporter gene directs expression of Turk E, Wright EM (1997) Membrane topology motifs in the SGLT cotransporter family. J Membr Biol 159: 1–20[CrossRef][Web of Science][Medline] Uldry M, Ibberson M, Horisberger JD, Chatton JY, Riederer BM, Thorens B (2001) Identification of a mammalian H+-myo-inositol symporter expressed predominantly in the brain. EMBO J 20: 4467–4477[CrossRef][Web of Science][Medline] Watari J, Kobae Y, Yamaki S, Yamada K, Toyofuku K, Tabuchi T, Shiratake K (2004) Identification of sorbitol transporters expressed in the phloem of apple source leaves. Plant Cell Physiol 45: 1032–1041 Weber A, Servaites JC, Geiger DR, Kofler H, Hille D, Groner F, Hebbeker U, Flügge UI (2000) Identification, purification, and molecular cloning of a putative plastidic glucose translocator. Plant Cell 12: 787–802 Wrubel W, Stochaj U, Sonnewald U, Theres C, Ehring R (1990) Reconstitution of an active lactose carrier in vivo by simultaneous synthesis of two complementary protein fragments. J Bacteriol 172: 5374–5381 This article has been cited by other articles:
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ABSTRACT
RESULTS
-glucuronidase (GUS) and with AtINT4 promoter-green fluorescent protein (GFP) reporter lines with immunohistochemical techniques and in analyses of a T-DNA insertion line. 
4; data not shown). Only pinitol, a methylated derivative of myoinositol, is also transported by AtINT4, although at lower rates (28.4% ± 2.1% [n = 12]) than myoinositol (
AtINT4-GP) or in two rabbits (
1,038 bp]) and partial (5' = mRNA fragment upstream from the T-DNA insertion site [primers: int4-5' and int4-840r 
-factor-invertase) gene fusion for study of protein localisation and gene expression in yeast. Proc Natl Acad Sci USA 80: 7080–7084
