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First published online October 19, 2007; 10.1104/pp.107.109033 Plant Physiology 145:1395-1407 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Arabidopsis INOSITOL TRANSPORTER2 Mediates H+ Symport of Different Inositol Epimers and Derivatives across the Plasma Membrane1,[C],[OA]Molekulare Pflanzenphysiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, D–91058 Erlangen, Germany
Of the four genes of the Arabidopsis (Arabidopsis thaliana) INOSITOL TRANSPORTER family (AtINT family) so far only AtINT4 has been described. Here we present the characterization of AtINT2 and AtINT3. cDNA sequencing revealed that the AtINT3 gene is incorrectly spliced and encodes a truncated protein of only 182 amino acids with four transmembrane helices. In contrast, AtINT2 codes for a functional transporter. AtINT2 localization in the plasma membrane was demonstrated by transient expression of an AtINT2-GREEN FLUORESCENT PROTEIN fusion in Arabidopsis and tobacco (Nicotiana tabacum) epidermis cells and in Arabidopsis protoplasts. Its functional and kinetic properties were determined by expression in yeast (Saccharomyces cerevisiae) cells and Xenopus laevis oocytes. Expression of AtINT2 in a  itr1 (inositol uptake)/ino1 (inositol biosynthesis) double mutant of bakers' yeast complemented the deficiency of this mutant to grow on low concentrations of myoinositol. In oocytes, AtINT2 mediated the symport of H+ and several inositol epimers, such as myoinositol, scylloinositol, D-chiroinositol, and mucoinositol. The preference for individual epimers differed from that found for AtINT4. Moreover, AtINT2 has a lower affinity for myoinositol (Km = 0.7–1.0 mM) than AtINT4 (Km = 0.24 mM), and the Km is slightly voltage dependent, which was not observed for AtINT4. Organ and tissue specificity of AtINT2 expression was analyzed in AtINT2 promoter/reporter gene plants and showed weak expression in the anther tapetum, the vasculature, and the leaf mesophyll. A T-DNA insertion line (Atint2.1) and an Atint2.1/Atint4.2 double mutant were analyzed under different growth conditions. The physiological roles of AtINT2 are discussed.
Myoinositol and its derivatives are central to numerous metabolic pathways under different physiological conditions. Myoinositol is a precursor in the biosynthesis of UDP-GlcUA, GalUA, Xyl, apiose, and Ara (Loewus and Murthy, 2000  ; Kanter et al., 2005), it is used for the formation of galactinol, a myoinositol-linked, activated form of Gal that is used for the biosynthesis of raffinose and its derivatives (Kandler and Hopf, 1982), and it is thought to be an initial substrate in the biosynthesis of L-ascorbic acid (Lorence et al., 2004). It may be conjugated to auxins to prevent their biological activity and to allow long-distance transport (Cohen and Bandurski, 1982), and in phospholipids (Lehle, 1990) or glycosylphosphatidylinositol-membrane anchors (Schultz et al., 1998) myoinositol provides the structural basis for membranes and membrane-attached proteins. Inositol-1,4,5-triphosphate plays an important role as second messenger, and myoinositol-1,2,3,4,5,6-hexakisphosphate (phytate) is used for the storage of inositol, phosphorus, and minerals (Shi et al., 2005), or as a structural component of proteins (Macbeth et al., 2005; Tan et al., 2007). Finally, myoinositol may represent a cellular energy currency (Raboy, 2003).
Living cells possess different inositol epimers, the most frequent ones being scylloinositol and D- or L-chiroinositol, and all of these epimers are found in phosphatidyl inositols (Narasimhan et al., 1997
The first cDNAs of putative plant myoinositol transporters, MITR1 and MITR2, were cloned from ice plants (Mesembryanthemum crystallinum), and MITR1-expressing yeast (Saccharomyces cerevisiae) cells were able to grow on lower extracellular concentrations of myoinositol than control cells (Chauhan et al., 2000 Here we present the molecular cloning of an AtINT2 cDNA (At1g30220), analyses of the tissue-specific expression of the AtINT2 gene, and the functional characterization and subcellular localization of the AtINT2 protein. Like AtINT4, AtINT2 is a plasma membrane-localized H+/inositol symporter. Expression of an AtINT2 cDNA in yeast and in Xenopus oocytes, however, revealed significant differences between the kinetic properties and substrate specificities of these transporters. Moreover, analyses of AtINT2 promoter/GUS and AtINT2 promoter/GFP reporter lines showed an expression pattern that differed from that of AtINT4. Finally, Atint2 mutant lines were studied. The putative physiological roles of AtINT2 are discussed.
Cloning of AtINT2 and AtINT3 cDNAs The AtINT3 gene (At2g35740) is predicted to encode a protein of 580 amino acids (NP_181117) that is similar to AtINT2 and AtINT4 with 60.4% and 63.4% identical amino acids, respectively. However, we did not succeed in amplifying an AtINT3 cDNA by reverse transcription (RT)-PCR. To overcome this problem we generated AtINT3 overexpressing plant material by infiltration of tobacco (Nicotiana tabacum) leaves with an Agrobacterium tumefaciens strain carrying the plasmid pPU6. The same strain was used for stable transformation of Arabidopsis. pPU6 drives expression of a genomic AtINT3 sequence (from start to stop including two predicted introns) under the control of the 35S promoter. RT-PCR reactions performed with total RNA from infiltrated tobacco leaves or transformed Arabidopsis plants were successful. Unexpectedly, in all cDNAs analyzed the first predicted AtINT3 intron was spliced incorrectly, i.e. 95 bp from the 3' end of the first predicted exon were removed together with the first intron, and in none of the analyzed sequences the second intron was spliced out. The obtained AtINT3 cDNAs were 1,742 bp long and carried a stop codon after the first 546 bp. This open reading frame (ORF) encodes a truncated protein with 182 amino acids. Only the first 148 amino acids (covering four of the predicted 12 transmembrane helices of AtINT3) corresponded to the predicted AtINT3 protein sequence. Amino acids 149 to 182 resulted from a frameshift due to the incorrect splicing. The obtained cDNA sequence was deposited in the EMBL database (accession no. AM778029). Obviously, neither in tobacco nor in Arabidopsis the genomic AtINT3 sequence is spliced as predicted, indicating that AtINT3 is most likely not a functional gene. Therefore, additional expression analyses in yeast and studies of the activity of the AtINT3 promoter were not performed.
The AtINT2 ORF (At1g30220) encodes a protein of 580 amino acids, with two consensus sequences for N-glycosylation (Asn310 and Asn382) and 12 predicted transmembrane helices (Schneider et al., 2006
Expression of the AtINT2 cDNA in Yeast
In a first approach we tried to determine the functional properties of AtINT2 in the bakers' yeast mutant D458-1B (Nikawa et al., 1991 The transformed yeast lines SSY38 (carrying the empty NEV-N-Leu vector), SSY39 (carrying the AtINT2 sense plasmid pSS51s), and SSY40 (antisense AtINT2) were grown on petri plates containing low (2 µg/mL) or high concentrations (20 µg/mL) of myoinositol to visualize a possible complementation of the described mutations. Figure 2 shows that SSY39 cells had, in fact, regained the capacity to grow on low myoinositol. As expected, no growth was seen for the control strains SSY38 and SSY40 on the same medium. After this initial evidence that AtINT2 might encode a myoinositol transporter, we tried to characterize the functional and kinetic properties in detail by transport analyses with 3H-labeled myoinositol in SSY39 cells. However, the uptake rates were too low to yield statistically solid data (data not shown). This suggested that the amount of AtINT2 protein in yeast plasma membranes was sufficient for complementation, but not high enough to allow direct measurements of radiolabeled substrates.
Expression of the AtINT2 cDNA in Xenopus laevis Oocytes
Xenopus laevis oocytes have been used for the successful expression of AtINT4 (Schneider et al., 2006 Figure 3A shows a typical recording of inward currents in the presence of various candidate substrates. Analyses were performed with 20-mM solutions of myoinositol, xylitol, mannitol, sorbitol, Glc, Fru, and Suc at an extracellular pH of 5.5. Only application of myoinositol resulted in strong inward currents. Much weaker currents (<10% of myoinositol) were elicited by Glc; no currents were obtained with any of the other compounds (quantitative analyses are shown in Fig. 4A ). This demonstrated that, as predicted from the analyses in yeast, AtINT2 does mediate the uptake of myoinositol. Furthermore, the obtained currents confirmed that AtINT2-driven transport is energy dependent, they suggested that a positive charge is likely to be symported with myoinositol, and they demonstrated that AtINT2 is quite specific and does not or hardly transport linear polyols, mono-, or disaccharides.
Myoinositol represents only one of nine possible inositol epimers. Seven of these epimers (allo-, D-chiro-, L-chiro-, muco-, myo-, neo-, and scylloinositol) are found in living cells, the others (cis- and epiinositol) possibly exist only as synthetically prepared compounds. Especially D-chiroinositol (altered orientation of the OH group at position 1 of myoinositol) and scylloinositol (altered orientation of the OH group at position 2 of myoinositol) were also found in plant phospholipids (Chien et al., 1996 ; Narasimhan et al., 1997; Carstensen et al., 1999). We compared the ability of AtINT2 to transport different epimers of inositol, including the frequently found epimers myo-, D-chiro-, and scylloinositol and two epimers that were not or hardly detected in plant cells (alloinositol [altered orientation of the OH group at position 6 of myoinositol] and mucoinositol [altered orientations of the OH groups at positions 1 and 6 of myoinositol]). Figures 3B (original recording) and 4B (quantitative analysis) demonstrate that in addition to the already shown transport of myoinositol (Figs. 3A and 4A) AtINT2 does also catalyze the transport of scyllo- and D-chiroinositol with only slightly reduced rates (90% and 70%, respectively). Lower current amplitudes (35% and 10%) were obtained in the presence of muco- and alloinositol. We also analyzed the myoinositol derivatives pinitol (monomethylated chiroinositol), phytate (6-fold phosphorylated), and galactinol (inositol attached to Gal). While pinitol was transported by AtINT2 (20% of the rate of myoinositol), no currents were elicited by phytate and galactinol (Figs. 3B and 4B).
The epimer specificity has so far neither been determined for the previously characterized AtINT4 transporter (Schneider et al., 2006 The identity of the cotransported ion was determined in the presence of different cations in the extracellular solution. There was no difference between the substrate-induced currents elicited in a buffer containing 110 mM NaCl and 2.5 mM KCl or in a buffer that replaced these salts by N-methylglucamine chloride (112.5 mM; Fig. 3C; n = 6). This demonstrates that neither K+ nor Na+ ions are symported, and that AtINT2 is most likely an inositol/H+ symporter. Figure 4C shows the pH dependence of inward H+ currents elicited by myoinositol in AtINT2-expressing Xenopus oocytes. AtINT2 shows highest transport rates at physiological and acidic pH values. At more alkaline values, myoinositol-induced currents decrease rapidly, which can be taken as additional, though circumstantial evidence that protons are the cosubstrate of AtINT2.
Finally, the Km value of AtINT2 was determined for myoinositol. Figure 4D shows Michaelis-Menten kinetics determined at a membrane potential (
For analyses of the tissue specificity of AtINT2 expression we generated and analyzed AtINT2 promoter/GUS plants and AtINT2 promoter/GFP plants. To this end a 1,448-bp promoter fragment was used to drive expression of GUS or GFP in plants that had been selected for BASTA resistance after transformation with the plasmids pLEX111 (=AtINT2 promoter/GUS) or pLEX106 (=AtINT2 promoter/GFP).
We obtained numerous transformants with both constructs and analyzed 30 independent AtINT2 promoter/GUS lines and 24 independent AtINT2 promoter-GFP lines. In none of the AtINT2 promoter/GFP lines we were able to detect GFP fluorescence, suggesting that the activity of the AtINT2 promoter is rather weak. This was confirmed by analyses of AtINT2 promoter/GUS plants. Although all plants showed the same tissue specificity of the AtINT2 promoter, the obtained GUS staining was weak and in many plants it was detected only after prolonged staining. The results are summarized in Figure 5
. Strongest GUS staining was detected in anthers (Fig. 5A). Analyses at higher magnification (Fig. 5B) and in destroyed anther tissue (Fig. 5C) revealed that this anther-specific GUS staining is not localized in pollen, but rather in a cell layer of the anther wall, most likely the tapetum. This is deduced from two observations: (1) the outermost cell layer of intact anthers shown in Figure 5B were not stained, and (2) individual pollen grains in squashed anther tissue did not show any GUS staining. Interestingly, the previously reported GUS staining in anthers of AtINT4 promoter/GUS plants (Schneider et al., 2006
AtINT2 promoter-dependent GUS activity was also seen in Arabidopsis leaves (Fig. 5D) and very weakly in roots (data not shown). Clearly, stronger staining was obtained in leaf vascular tissue of AtINT2 promoter-GUS plants, however, staining was also seen in all other parts of the leaves.
Antisera were raised in one guinea pig and two rabbits against a 26-amino acid peptide from the very C terminus of AtINT2. This sequence is not found in any other Arabidopsis protein and does not show significant homology to the C termini of the other AtINT proteins. The quality of the obtained sera was tested on detergent extracts from total membranes isolated from yeast strains ScLEX41 (SEY2102 [Emr et al., 1983
The difference between the apparent molecular mass (55 kD) and the molecular mass predicted from the DNA sequence (63.4 kD) was not unexpected. Typically, lipophilic proteins run at lower apparent molecular masses on SDS gels. This unusual running behavior results from excess binding of SDS of highly lipophilic proteins (Beyreuther et al., 1980 ; Gahrtz et al., 1994; Barth et al., 2003; Schneider et al., 2006).
Next we tested the quality of the antisera on thin sections of yeast cells that expressed AtINT2 either in sense (SSY39) or in antisense orientation (SSY40; see above). Sections of these cells were prepared using the identical protocol usually applied for the fixation and embedding of plant material (Meyer et al., 2004
Therefore, affinity-purified
The subcellular localization of AtINT2 in planta was analyzed using an AtINT2 protein with GFP fused to its C terminus. To this end, the plasmid pMG002 that drives expression of the AtINT2-GFP fusion under the control of an enhanced 35S promoter was used for transient expression in Arabidopsis protoplasts (Figs. 7A ) or in particle bombarded epidermis cells of Arabidopsis (Fig. 7B) or tobacco (Fig. 7C). Transformed cells and protoplasts were analyzed by confocal microscopy. In all analyses the red autofluorescence of the chloroplasts was localized inside the GFP-labeled structure (arrows in Fig. 7). No GFP fluorescence was found in any other structure inside the transformed cells, indicating that in both plant expression systems (Arabidopsis and tobacco) the AtINT2-GFP fusion protein is located in the plasma membrane.
Analysis of an Atint2 Mutant Line Screening of publicly accessible libraries identified a mutant line (GARLIC_1264A7 = Atint2.1) with a T-DNA insertion in the second intron of the AtINT2 gene, 644 bp after the start ATG (Fig. 8A ). We performed PCR reactions to identify homozygous Atint2.1 plants (e.g. plant 2 in the PCR shown in Fig. 8A) and used these plants for further analyses. Homozygous mutant plants showed a complete loss of intact AtINT2 mRNA (1,742-bp band in Fig. 8B). Partial AtINT2 mRNAs, however, representing sequences flanking the T-DNA insertion could be amplified also from Atint2.1 RNA preparations. The 197-bp fragment (N in Fig. 8B) flanking the predicted right border of the T-DNA insertion (RB in Fig. 8A) is likely to result from AtINT2 promoter activity. The 240-bp fragment (C in Fig. 8B) flanking the identified left border (Fig. 8A) of the T-DNA insertion most likely results from promoter activity within the T-DNA insertion. Growth analyses of homozygous Atint2.1 plants 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), in the presence of mannitol, or on different concentrations of myoinositol in the growth medium (0–100 mM) revealed no visible or metabolic (e.g. altered sugar, polyol, or cyclitol concentrations; data not shown) 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 AtINT2.
Analysis of an Atint2.1/Atint4.2 Double Mutant
A possible reason for the lack of a detectable phenotype in the Atint2.1 mutant might be a functional complementation of the defective Atint2.1 allele by AtINT4. AtINT4 promoter/GUS and AtINT4 promoter/GFP analyses (Schneider et al., 2006 For the identification of Atint2.1/Atint4.2 double mutants, comparative PCRs were performed with genomic DNA isolated from Columbia-0 (Col-0) plants or from potential double mutants and a set of three primers that allowed simultaneous amplification of fragments from wild-type and mutant alleles in a single PCR reaction. Figure 8C shows the characterization of a homozygous Atint2.1/Atint4.2 double mutant that yielded only PCR fragments of the two mutant alleles. These fragments were not amplified from Col-0 DNA that showed the expected wild-type fragments for both genes. Like the Atint2.1 single mutant, the Atint2.1/Atint4.2 double mutant neither developed a growth phenotype under different growth conditions nor did we observe differences in the carbohydrate compositions of leaf extracts (myoinositol, Glc, Fru, Suc; data not shown).
This article presents a detailed characterization of AtINT2 as a plasma membrane-localized H+ symporter with medium affinity for various inositol epimers, and of AtINT3 as a gene that very likely encodes a truncated protein. AtINT2 and AtINT3 represent two of four predicted inositol transporter genes (AtINT1 to AtINT4) that form a subfamily within the MST-like superfamily of Arabidopsis that was named after the AtSTP family of plasma membrane-localized monosaccharide transporters (Sauer et al., 1990 ; Büttner and Sauer, 2000; Büttner, 2007).
So far, less than 50% of the members of the MST-like superfamily (53 genes in Arabidopsis) have been characterized on a functional basis. This includes most members of the STP subfamily (Büttner, 2007
Our attempts to obtain an AtINT3 cDNA via RT-PCR from total RNA from Arabidopsis rosettes failed repeatedly. Moreover, publicly accessible libraries did not contain AtINT3 cDNA clones. Finally, no correctly spliced AtINT3 cDNAs could be isolated from plant tissues (Arabidopsis or tobacco) that expressed genomic AtINT3 sequences under the control of the 35S promoter. All obtained cDNAs were incorrectly spliced and encoded only a truncated protein. As can be seen from the phylogenetic tree shown in Figure 1, AtINT3 is closely related to AtINT4, suggesting that the corresponding genes may have duplicated in the more recent past of Arabidopsis evolution. Obviously one of the duplicated genes, AtINT3, accumulated mutations that eventually caused a loss of function. Similar observations have been reported for members of the Arabidopsis Suc transporter family (Sauer et al., 2004
The successful functional analyses in yeast (complementation of a defect in the plasma membrane myoinositol transporter Itr1p) and Xenopus suggested that AtINT2 might be a plasma membrane-localized protein in plant cells. This was confirmed by transient expression analyses of an AtINT2-GFP construct that resulted in GFP fluorescence exclusively in the plasma membrane of transformed cells (Fig. 7). Moreover, the Xenopus data characterized AtINT2 as an H+/inositol symporter with medium affinity to myoinositol. In contrast to AtINT4 that has a 4-fold lower, Another difference between AtINT2 and AtINT4 was observed, when the currents elicited by different inositol epimers were compared (Figs. 3B and 4B). At most likely saturating substrate concentrations (20 mM), AtINT2 catalyzed the uptake of myoinositol and scylloinositol with similar rates. D-chiro-, muco-, and alloinositol were also transported, although with decreasing uptake rates. Under identical conditions, however, AtINT4 preferred myoinositol and D-chiroinositol (D-chiroinositol even slightly better), transported scylloinositol with significantly lower rates, and muco- and alloinositol turned out to be only poor substrates for AtINT4. This demonstrates that AtINT2 and AtINT4 respond differently to altered orientations of the OH groups at position 1 of myoinositol (as in chiroinositol; best substrate for AtINT4) and position 2 (as in scylloinositol; very good substrate for AtINT2). In contrast, an altered orientation of the OH group at position 6 of myoinositol (as in muco- and alloinositol) resulted in reduced transport by both proteins.
AtINT2 and AtINT4 transported pinitol, the monomethylated derivative of chiroinositol, only with reduced rates (about 20%–30% of chiroinositol; Fig. 4B; Schneider et al., 2006
Although data on tissue or cellular concentrations of scyllo- and chiroinositol are not available for Arabidopsis, the finding of these inositol epimers in numerous other plant species (Kinnard et al., 1995
Substrate specificities of inositol transporters have also been studied in several nonplant organisms, and for some of these transport systems they are similar to those described for AtINT2 and AtINT4. For the ciliate Tetrahymena vorax, for example, it was found that the most frequently occurring epimers myo-, scyllo-, and D-chiroinositol were transported across the plasma membrane (Kersting and Ryals, 2004
Analyses of the protein sequences of the (putative) inositol transporters shown in the phylogenetic tree in Figure 1 revealed that the loops between the predicted transmembrane helices IX and X of all transporters in clades 2 and 3 are about 80 amino acids longer than the respective loops of the proteins in clade 1. These additional 80 amino acids possess an unusually large number of highly conserved Cys residues. Four of these residues are part of two Cys-X-X-Cys motifs typically separated by only few amino acids (e.g. 399-CMTCLKASSPSCGYC-413 in AtINT2 and 406-CMKCLRSECGFC-417 in AtINT4). Since AtINT2 and AtINT4 were both localized to the plasma membrane, their Cys-X-X-Cys-containing loops are predicted to face the extracellular space and the lumen of the endoplasmic reticulum during biosynthesis and targeting.
Possibly, these highly conserved Cys residues are substrates for endoplasmic protein disulphide isomerases. Alternatively, the Cys-X-X-Cys motifs may provide some yet uncharacterized function to the extracellular loop or confer special redox sensitivity to the protein. Thioredoxins, for example, and bacterial disulphide isomerases have quite similar Cys-X-X-Cys motifs (Elton et al., 2005
Expression of the AtINT2 gene was observed in anthers (Fig. 5, A–C), in the vascular tissue (Fig. 5D), and to a lesser extent also in the leaf mesophyll (Fig. 5D). The low intensity of the obtained GUS staining and the lack of GFP fluorescence suggested low expression levels for AtINT2, and, in fact, we were not able to immunolocalize AtINT2 protein in plant tissue, although the antiserum was shown to work on western blots (Fig. 6A) and in thin sections of AtINT2-expressing yeast cells.
At first sight, the expression patterns of AtINT2 (Fig. 5) and AtINT4 (Schneider et al., 2006 More detailed analyses of the GUS data from AtINT2 and AtINT4 promoter/GUS plants revealed also differences in the expression patterns in leaves. Whereas the AtINT2 promoter is active in the vasculature and to a lesser extent also in all other parts of the leaf (Fig. 5D), the activity of the AtINT4 promoter is strictly confined to the vasculature.
Due to the absence of a phenotypic difference between Atint2.1 single or Atint2.1/Atint4.2 double mutants and wild-type plants, a more detailed prediction of the physiological role(s) of AtINT2 is difficult. In the vasculature it may be involved in the supply of inositol for galactinol and thus for raffinose biosynthesis. The concentrations of raffinose in Arabidopsis, however, are very low (Haritatos et al., 2000 One may speculate that triple mutants that include also a T-DNA insertion in the AtINT1 gene might provide additional information. However, only recently AtINT1 has been characterized as a tonoplast-localized inositol transporter, and Atint1 mutants show increased cellular inositol concentrations and reduced root development (S. Schneider and N. Sauer, unpublished data). The construction of an Atint1/Atint2/Atint4 triple mutant is on the way, however, with the background of the observed Atint1-based phenotypic differences additional effects of Atint2 or Atint4 mutations will be difficult to identify.
Strains and Growth Conditions
Arabidopsis (Arabidopsis thaliana; Col wild type) plants were grown in growth chambers on potting soil under a 16 h light/8 h dark regime (22°C, 60% relative humidity) or in the greenhouse under ambient conditions. For expression of AtINT2 cDNAs in yeast (Saccharomyces cerevisiae) we used strains D458-1B (Nikawa et al., 1991
AtINT3 cDNA was amplified from tobacco (Nicotiana tabacum) plants infiltrated with Agrobacteria carrying plasmid pPU6 or from Arabidopsis plants stably transformed (Clough and Bent, 1998 After isolation of total RNA (RNeasy plant mini kit; Qiagen) from infiltrated tobacco leaves (24 h after infiltration) or from rosette leaves of BASTA-selected Arabidopsis plants, AtINT3 cDNA was amplified using the primers AtINT3g + 1f and AtINT3g + 1926r, cloned into pCR-Blunt-II-Topo (Invitrogen), and sequenced.
PCR-based cloning of the AtINT2 cDNA was described (Schneider et al., 2006
Oocytes were injected (General Valve Picospritzer III, Parker Hannifin Corp.) with cRNA from AtINT2 or AtINT4 or with RNase-free water and stored at 16°C in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, adjusted to pH 7.4 with NaOH) supplemented with 1% penicillin-streptomycin (Sigma) or Barth's solution [88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 82 mM MgCl2, 2.4 mM NaHCO3, 10 mM HEPES, adjusted to pH 7.6 with NaOH] supplemented with 0.1% gentamycin (Sigma). Oocyte currents were studied 4 to 6 d after injection with the two-electrode voltage-clamp technique (Hedrich et al., 1995
A 1,448-bp AtINT2 promoter fragment was PCR amplified from genomic Arabidopsis DNA. The primers (INT2-p5: 5'-ATAACTTTAAGCTTTTTGTTAGAA-3'; INT2-p3: 5'-TCCTCCCTCCATGGTTTTTTGGGTTAAAAGAGTTAGAA-3') introduced an N-terminal HindIII and a C-terminal NcoI site that were used to clone the fragment in front of the ORF of GFP and a transcriptional terminator in a pUC19-based plasmid (pEPS1/pUC19; Imlau et al., 1999
The AtINT2 coding sequence was PCR amplified using the primers AtINT2-5-BspH (5'-TCATGAAGGGAGGAATAATACATG-3') and AtINT2-5-bspH (5'-TCATGACTGCACTCTGGTTTTGTTTCTC-3'). These primers introduced BspHI sites at the start and at the very end of the AtINT2 ORF, thereby replacing the stop codon of the original AtINT2 sequence. This modified AtINT2 ORF was inserted into the unique NcoI cloning site representing the start ATG of the GFP ORF in the pSO35e plasmid (Klepek et al., 2005
pMG002 was used for transient expression of AtINT2 in Arabidopsis protoplasts (polyethylene glycol transformation; modified after Abel and Theologis, 1994
The T-DNA insertion line Atint2.1 (GARLIC_1264A7 = SAIL_1264_A07) was identified using the Salk Institute T-DNA Express gene-mapping tool (Alonso et al., 2003 Primers Int2cs5 (5'-ATATCTCTGCGGCCGCAAAAATGGAGGGAGGAATAATACAT-3'), Int2cs3 (5'-ATATCTCTGCGGCCGCTCATGCACTCTGGTTTTGTTTCTCA-3'), Int2-1/108, and Int2-1/1226r were used for RT-PCRs with total RNA from wild-type and mutant Arabidopsis leaves. Primers AtACT2g + 846f (5'-ATTCAGATGCCCAGAAGTCTTGTT-3') and AtACT2g + 1295r (5'-GAAACATTTTCTGTGAACGATTCCT-3') were used to amplify the AtACT2 mRNA.
For
Binding of
Protein extracts of total membrane fractions from bakers' yeast were prepared as described (Sauer and Stolz, 2000
Concentrations of sugars and sugar alcohols were determined with an ICS-3000 system (Dionex) with a gradient pump (ICS-3000 SP), a degaser module, an autosampler (ICS-3000 AS), and a pulsed amperometric detector (ICS-3000 DC). Anionic compounds were separated on 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 that was placed between the eluent and separation columns to remove anionic contaminants in the eluents. As eluent, 612 mM sodium hydroxide made from purest water (Millipore) and 50% NaOH (Fluka) was used. The column was equilibrated at a flow rate of 0.4 mL min–1. The duration of the run was 60 min. Calibration and quantitative calculation of carbohydrates was performed with the Dionex chromeleon software 6.7.
Images of GFP fluorescence were made with an epifluorescence microscope (Zeiss Axioskop, Carl Zeiss Jena GmbH; excitation wavelength 460–500 nm) or with a confocal laser-scanning microscope (Leica TCS SPII, Leica Microsystems). Emitted fluorescence was monitored at detection wavelengths longer than 510 nm. Confocal images were processed using the Leica Confocal Software 2.5 (Leica Microsystems). GUS plants were analyzed using a stereomicroscope (Leica MZFLIII; Leica Microsystems) or a microscope (Zeiss Axioskop, Carl Zeiss Jena GmbH). Images were processed using analySIS Doku 3.2 Software (Soft Imaging System). Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AM778029 for AtINT3.
We thank Ruth Stadler for experimental help and Angelika Wolf for growing the Arabidopsis plants. We are grateful to Christoph Korbmacher for the frequent and generous supply with Xenopus laevis oocytes. Received September 13, 2007; accepted September 24, 2007; published October 19, 2007.
1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Arabidopsis Functional Genomics Network; grant no. Sa 382/13–1 to N.S.).  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).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.109033 * Corresponding author; e-mail nsauer{at}biologie.uni-erlangen.de.
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 Aluri S, Büttner M (2007) Identification and functional expression of the Arabidopsis thaliana vacuolar glucose transporter 1 and its role in seed germination and flowering. Proc Natl Acad Sci USA 104: 2537–2542 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, 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 HW, 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 Büttner M (2007) The monosaccharide transporter(-like) gene family in Arabidopsis. FEBS Lett 581: 2318–2324[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] Carpaneto A, Geiger D, Bamberg E, Sauer N, Fromm J, Hedrich R (2005) Phloem-localized, proton-coupled sucrose carrier ZmSUT1 mediates sucrose efflux under control of sucrose gradient and pmf. J Biol Chem 280: 21437–21443 Carstensen S, Pliska-Matyshak G, Bhuvarahamurthy N, Robbins KM, Murthy PP (1999) Biosynthesis and localization of phosphatidyl-scyllo-inositol in barley aleurone cells. Lipids 34: 67–73[Medline] Chauhan S, Forsthoefel N, Ran Y, Quigley F, Nelson DE, Bohnert HJ (2000) Na+/myo-inositol symporters and Na+/H+-antiport in Mesembryanthemum crystallinum. Plant J 24: 511–522[CrossRef][Web of Science][Medline] Chien CT, Lin TP, Juo CG, Her GR (1996) Occurance of a novel galactopinitol and its changes with other non-reducing sugars during development of Leucaena laucocephala seeds. Plant Cell Physiol 37: 539–544 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] Cohen JD, Bandurski RS (1982) Chemistry and physiology of the bound auxins. Annu Rev Plant Physiol 33: 403–430[Web of Science] Dunn SD (1986) Effects of the modification of transfer buffer composition and 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] Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard CS (2006) Gateway-compatible vectors for plant functional genomics and proteomics. Plant J 45: 616–629[CrossRef][Web of Science][Medline] Elton TC, Holland SJ, Frost LS, Hazes B (2005) F-like type IV secretion systems encode proteins with thioredoxin folds that are putative DsbC homologues. J Bacteriol 187: 8267–8277 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] 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, Ayre BG, Turgeon R (2000) Identification of phloem involved in assimilate loading in leaves by activity of the galactinol synthase promoter. Plant Physiol 123: 929–937 Hasegawa PM, Bressan RA, Zhu JK, Bohnert H (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol 51: 463–499[CrossRef][Web of Science][Medline] Hedrich R, Moran O, Conti F, Busch H, Becker D, Gambale F, Dreyer I, Kuch A, Neuwinger K, Palme K (1995) Inward rectifier potassium channels in plants differ from their animal counterparts in response to voltage and channel modulators. Eur Biophys J 24: 107–115[Web of Science][Medline] 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] Ichimura K, Kohata K, Yamaguchi Y, Douzono M, Ikeda H, Koketsu M (2000) Identification of L-inositol and scyllitol and their distribution in various organs in Chrysanthemum. Biosci Biotechnol Biochem 64: 865–868[CrossRef][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] Jin JH, Seyfang A (2003) High-affinity myo-inositol transport in Candida albicans: substrate specificity and pharmacology. Microbiology 149: 3371–3381 Kandler O, Hopf H (1982) Oligosaccharides based on sucrose (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] Kersting M, Ryals PE (2004) Sodium-dependent transport of [3H](1D)chiro-inositol by Tetrahymena. J Eukaryot Microbiol 51: 307–311[CrossRef][Web of Science][Medline] Kinnard RL, Narasimhan B, Pliska-Matyshak G, Murthy PP (1995) Characterization of scyllo-inositol-containing phosphatidylinositol in plant cells. Biochem Biophys Res Commun 210: 549–555[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] 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 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] Loewus FA (1990) Structure and occurrence of inositols in plants. In DJ Morré, WF Boss, FA Loewus, eds, Inositol Metabolism in Plants. Wiley-Liss Inc, New York, pp 1–11 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 Macbeth MR, Schubert HL, VanDemark AP, Lingam AT, Hill CP, Bass BL (2005) Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science 309: 1534–1539 Meyer S, Lauterbach C, Niedermeier M, Barth I, Sjolund RD, Sauer N (2004) Wounding enhances expression of AtSUC3, a sucrose transporter from Arabidopsis sieve elements and sink tissues. Plant Physiol 134: 684–693 Miyazaki S, Rice M, Quigley F, Bohnert HJ (2004) Expression of plant inositol transporters in yeast. Plant Sci 166: 245–252 Mongan TP, Ganapasam S, Hobbs SB, Seyfang A (2004) Substrate specificity of the Leishmania donovani myo-inositol transporter: critical role of inositol C-2, C-3 and C-5 hydroxyl groups. Mol Biochem Parasitol 135: 133–141[CrossRef][Web of Science][Medline] Murakeözy EP, Nagy Z, Duhazé C, Bouchereau A, Tuba Z (2003) Seasonal changes in the levels of compatible osmolytes in three halophytic species of inland saline vegetation in Hungary. J Plant Physiol 160: 395–401[CrossRef][Web of Science][Medline] Narasimhan B, Pliska-Matyshak G, Kinnard R, Carstensen S, Ritter MA, van Weymarn L, Murthy PN (1997) Novel phosphoinositides in barley aleurone cells. Plant Physiol 113: 1385–1393[Abstract] 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 Ostlund RE Jr, Seemayer R, Gupta S, Kimmel R, Ostlund EL, Sherman WR (1996) A stereospecific myo-inositol/D-chiro-inositol transporter in HepG2 liver cells. J Biol Chem 271: 10073–10078 Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12: 357–358 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 (1990) Biochemistry and genetics of phytic acid biosynthesis. In DJ Morré, WF Boss, FA Loewus, eds, Inositol Metabolism in Plants. Wiley-Liss Inc, New York, pp 55–76 Raboy V (2003) myo-Inositol-1,2,3,4,5,6-hexakisphosphate. Phytochemistry 64: 1033–1043[CrossRef][Web of Science][Medline] Reinders A, Panshyshyn JA, Ward JM (2005) Analysis of transport activity of Arabidopsis sugar alcohol permease homolog AtPLT5. J Biol Chem 280: 1594–1602 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, Ludwig A, Knoblauch A, Rothe P, Gahrtz M, Klebl F (2004) AtSUC8 and AtSUC9 encode functional sucrose transporters, but the closely related AtSUC6 and AtSUC7 genes encode aberrant proteins in different Arabidopsis ecotypes. Plant J 40: 120–130[CrossRef][Web of Science][Medline] Sauer N, Stolz J (1994) SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's 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, Oxford, pp 79–105 Schneider S, Schneidereit A, Konrad KR, Hajirezaei M-R, Gramann M, Hedrich R, Sauer N (2006) Arabidopsis thaliana INOSITOL TRANSPORTER 4 mediates high affinity H+-transport of myoinositol across the plasma membrane. Plant Physiol 141: 565–577 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 L. 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] Smith AE, Phillips EV (1982) The maize low-phytic acid 3 encodes a myo-inositol kinase that plays a role in phytic acid biosynthesis in developing seeds. Physiol Plant 54: 31–33[CrossRef] Streeter JG, Lohnes DG, Fioritto RJ (2001) Patterns of pinitol accumulation in soybean plants and relationships to drought tolerance. Plant Cell Environ 24: 429–438[CrossRef] Tan X, Calderon-Villalobos LIA, Sharon M, Zheng C, Robinson CV, Estelle M, Zheng N (2007) Mechanism of auxin perception by the TIR1 ubiquitin ligase. Nature 446: 640–645[CrossRef][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 25: 4876–4882 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 Wormit A, Trentmann O, Feifer I, Lohr C, Tjaden J, Meyer S, Schmidt U, Martinoia E, Neuhaus HE (2006) Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport. Plant Cell 18: 3476–3490 This article has been cited by other articles:
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) of –60 mV and at an extracellular pH of 5.5. Comparison of the Km value calculated from this and similar analyses at different membrane potentials (insert in 
AtINT2-GP; similar results were obtained with the rabbit-derived antisera). A band with an apparent molecular mass of about 55 kD was detected only in extracts from AtINT2-expressing cells. This band was absent from extracts from SSY9 control cells showing that the labeled band in ScLEX41 cells represents the AtINT2 protein. 
-factor-invertase) gene fusion for study of protein localisation and gene expression in yeast. Proc Natl Acad Sci USA 80: 7080–7084
