Sulfate transporters present at the root surface facilitate uptake of sulfate from the environment. Here we report that uptake of sulfate at the outermost cell layers of Arabidopsis root is associated with the functions of highly and low-inducible sulfate transporters, Sultr1;1 and Sultr1;2, respectively. We have previously reported that Sultr1;1 is a high-affinity sulfate transporter expressed in root hairs, epidermal and cortical cells of Arabidopsis roots, and its expression is strongly upregulated in plants deprived of external sulfate. A novel sulfate transporter gene, Sultr1;2, identified on the BAC clone F28K19 of Arabidopsis, encoded a polypeptide of 653 amino acids that is 72.6% identical to Sultr1;1 and was able to restore sulfate uptake capacity of a yeast mutant lacking sulfate transporter genes (Km for sulfate = 6.9 ± 1.0 µm). Transgenic Arabidopsis plants expressing the fusion gene construct of the Sultr1;2 promoter and green fluorescent protein (GFP) showed specific localization of GFP in the root hairs, epidermal and cortical cells of roots, and in the guard cells of leaves, suggesting that Sultr1;2 may co-localize with Sultr1;1 in the same cell layers at the root surface. Sultr1;1 mRNA was abundantly expressed under low-sulfur conditions (50–100 µm sulfate), whereas Sultr1;2 mRNA accumulated constitutively at high levels under a wide range of sulfur conditions (50–1500 µm sulfate), indicating that Sultr1;2 is less responsive to changes in sulfur conditions. Addition of selenate to the medium increased the level of Sultr1;1 mRNA in parallel with a decrease in the internal sulfate pool in roots. The level of Sultr1;2 mRNA was not influenced under these conditions. Antisense plants of Sultr1;1 showed reduced accumulation of sulfate in roots, particularly in plants treated with selenate, suggesting that the inducible transporter Sultr1;1 contributes to the uptake of sulfate under stressed conditions.
Acquisition of sulfur can limit plant growth. The primary step of sulfur assimilation is the uptake of sulfate from the environment. Proton/sulfate co-transporters bound in the plasma membranes are responsible for specific absorption and symplastic movement of sulfate. Higher plants have multiple isoforms of sulfate transporters with different affinities, capacities and cell type-specific localization. These enable efficient uptake and internal translocation of sulfate throughout the whole plant. Following distribution to various organs through the vascular system, sulfate is converted to sulfur-containing amino acids and metabolites that are important for numerous biological reactions (Leustek and Saito, 1999; Saito, 2000).
The first isolation of higher plant cDNAs that encode sulfate transporters was reported from a tropical forage legume, Stylosanthes hamata, by phenotypic complementation of a yeast mutant lacking the gene encoding the high-affinity sulfate transporter SUL1 (Smith et al., 1995). In the past few years genes for sulfate transporters have been isolated and characterized from various plant species (Bolchi et al., 1999; Smith et al., 1997; Takahashi et al., 1996; Takahashi et al., 1997; Takahashi et al., 1999; Takahashi et al., 2000; Vidmar et al., 1999; Vidmar et al., 2000). These sulfate transporter proteins were classified into four groups based on the similarity of their protein sequences, kinetic properties and tissue-specific localization (Grossman and Takahashi, 2001). The members of each group are suggested to have specialized functions for the uptake and distribution of sulfate in plants. Members of group 1 encode high-affinity sulfate transporters that are primarily expressed in roots of sulfur-starved plants. It is suggested that these transporters mediate the initial uptake of sulfate from the soil when sulfate is limiting. Transporters in group 2 have lower affinity for sulfate and are expressed in vascular tissues, indicating their role in the internal translocation of sulfate in plants (Smith et al., 1995; Takahashi et al., 1997; Takahashi et al., 2000). The members of group 3 share significant sequence similarities and are expressed preferentially in leaves, but their roles have not been characterized in detail. Group 4 includes unique transporters that are specifically localized in the plastids and have similarity to putative sulfate transporter genes in algae (Takahashi et al., 1999).
Absorption of sulfate from the soil is the initial and essential step for acquisition of sulfur in plants. Sulfate uptake is tightly regulated by the external sulfur supply. The capacity for sulfate uptake increases during a period of sulfur limitation in plant roots (Clarkson et al., 1983; Smith et al., 1997; Vidmar et al., 2000). Previous studies on barley and Arabidopsis indicate that levels of transcripts corresponding to the HVST1 and Sultr1;1 high-affinity sulfate transporters are highly regulated by changes in the sulfur status of plants, and increase in parallel with sulfate uptake rates (Smith et al., 1997; Takahashi et al., 2000; Vidmar et al., 1999, Vidmar et al., 2000). During sulfur deprivation, specific expression of Sultr1;1 in the root hair, epidermis and cortex of roots may serve for scavenging a limiting amount of sulfate at the root/soil interface in Arabidopsis (Takahashi et al., 2000). In this study, we report the identification of a second isoform of group 1 high-affinity sulfate transporter, Sultr1;2, which may contribute to constitutive uptake of sulfate at the root surface of Arabidopsis. We demonstrate that induced accumulation of Sultr1;1 mRNA increases sulfate uptake capacity under sulfur-deficient conditions. Acquisition of sulfate from the environment is mediated by two high-affinity transporters, Sultr1;1 and Sultr1;2, which are differently regulated at mRNA levels by sulfur conditions.
Identification of Sultr1;2 in Arabidopsis
Previously we have reported that Arabidopsis has at least seven different sulfate transporter genes (Takahashi et al., 2000). Completion of the genome sequencing has revealed seven additional candidates that may encode sulfate transporters (The Arabidopsis Genome Initiative, 2000). Among these new members we found a homologue of the high-affinity sulfate transporter, Sultr1;1, and have designated this new member Sultr1;2. The putative open reading frame of Sultr1;2 was located directly adjacent to Sultr2;2 on BAC clone F28K19 (accession no. AC009243). The cDNA encoding Sultr1;2 was isolated by RT–PCR from the root RNA of 2-week-old Arabidopsis, and the cDNA sequence confirmed to be identical with the nucleotide sequence of the genome. The 5′ and 3′ ends of Sultr1;1 and Sultr1;2 mRNA were determined by RACE (rapid amplification of cDNA ends) methods (Figure 1a). Comparison of the nucleotide sequences of the full-length cDNA and the corresponding region of the BAC clone revealed that junction points of exons and introns in Sultr1;2 are well conserved with those in Sultr1;1 (Figure 1a). Consequently, Sultr1;2 encoded a 653 amino acid polypeptide that is highly homologous with Sultr1;1 from Arabidopsis (Takahashi et al., 2000; Vidmar et al., 2000) (Figure 1b). The deduced amino acid sequence of Sultr1;2 showed approximately 70% identity with other high-affinity sulfate transporters isolated from vascular plants (Smith et al., 1995; Smith et al., 1997; Vidmar et al., 1999).
Sultr1;2 encodes the high-affinity sulfate transporter
The sulfate uptake capacity of the Sultr1;2 encoded polypeptide was determined in the yeast mutant CP154-7A (Cherest et al., 1997). CP154-7A, having disruptions in both SUL1 and SUL2 high-affinity sulfate transporter genes, is unable to grow on medium containing less than 1 mm sulfate. The coding sequence of Sultr1;2 cDNA was cloned under the yeast glyceraldehyde-3-phosphate dehydrogenase promoter in the yeast expression vector pYE22m (Ashikari et al., 1989), and transformed into the mutant strain CP154-7A. Expression of Sultr1;2 rescued the growth of CP154-7A on medium containing 0.1 mm of sulfate as a sole sulfur source (Figure 2a). Sultr1;2 therefore encodes a sulfate transporter protein that is able to complement the functions of two sulfate transporters in the plasma membranes of yeast cells.
The kinetic properties of the Sultr1;2 transporter were determined by measuring sulfate uptake rates in complemented yeast cells. Yeast cells were suspended in a medium containing 35S-labelled sulfate, and incorporation of the radioactivity was assayed. Measurements of uptake rates at different sulfate concentrations indicated that sulfate uptake mediated by Sultr1;2 follows Michaelis–Menten kinetics. The calculated Km value of Sultr1;2 for sulfate was 6.9 ± 1.0 µm (Figure 2b), comparable with the Km of other high-affinity sulfate transporters in higher plants (Smith et al., 1995; Smith et al., 1997; Takahashi et al., 2000). These results indicate that Sultr1;2 functions as a high-affinity sulfate transporter capable of taking up sulfate from low external sulfate concentrations.
Sultr1;2 co-localizes with Sultr1;1 in Arabidopsis roots
Cell type-specific expression of Sultr1;2 was studied in transgenic Arabidopsis plants transformed with a fusion gene construct of Sultr1;2 promoter and green fluorescent protein (GFP). A DNA fragment flanking 2766 bp upstream of the translational initiation site of Sultr1;2 was fused to the coding sequence of GFP (Chiu et al., 1996), and introduced into Arabidopsis plants by Agrobacterium-mediated transformation (Clough and Bent, 1998). More than 10 independent transgenic lines were analysed to confirm expression of the GFP in specific cell types. In roots, GFP was accumulated in the epidermis (Figure 3a,b), cortex (Figure 3c,d) and root hairs (Figure 3e). GFP was preferentially expressed in the cortex of the mature part of roots. The cell types in which GFP was expressed in roots are identical to those in transgenic plants expressing the Sultr1;1 promoter–GFP fusion gene construct (Takahashi et al., 2000). These results suggest that two sulfate transporters, Sultr1;1 and Sultr1;2, may co-localize in the same cell layers at the root surface that are in contact with the soil solution. Weak fluorescence of GFP was found in the guard cells of the Sultr1;2–GFP plants (Figure 3f). The guard cell expression was not observed in the leaves of Sultr1;1–GFP plants.
Effects of sulfur conditions on mRNA levels of Sultr1;1 and Sultr1;2
The effects of sulfur limitation on steady-state mRNA levels of Sultr1;1 and Sultr1;2 were analysed by RT–PCR. Northern blot hybridization generally gave the same accumulation patterns of Sultr1;2 mRNA as shown in the RT–PCR experiments (data not shown). Sultr1;1 mRNA was detectable only by RT–PCR, suggesting a difference in the levels of mRNA abundance of these two high-affinity sulfate transporters.
To analyse the response of Sultr1;1 and Sultr1;2 expression to withdrawal of the external sulfate, 2-week-old Arabidopsis plants grown on the control sulfur-replete media (1500 µm sulfate) were transferred onto sulfur-deficient media containing no sulfate. The increase in roots of mRNA corresponding to Sultr1;1 by this treatment was generally the same as previously reported (Takahashi et al., 2000). Upon withdrawal of sulfate, Sultr1;1 mRNA increased continuously over 48 h to approximately 10 times the starting levels (Figure 4). However, mRNA corresponding to Sultr1;2 increased two- to threefold in the first 6 h in a similar manner to the increase in Sultr1;1 transcripts (Figure 4), but then remained relatively constant for the remainder of the 48 h period. Sultr1;1 and Sultr1;2 transcripts were detected at low levels in leaves, and levels of both were slightly enhanced by 48 h of sulfate starvation (Figure 4).
The effects of chronic sulfur limitation were investigated in Arabidopsis plants grown continuously for 2 weeks on media containing 50, 100, 200, 400, 800 or 1500 µm sulfate as a sole sulfur source. In roots, Sultr1;1 mRNA accumulated exclusively under low-sulfur conditions (Figure 5), as previously reported (Takahashi et al., 2000). The level of mRNA corresponding to Sultr1;1 in plants grown on 50 µm sulfate media was approximately 10 times higher than that in control plants grown on 1500 µm sulfate. However, the relative levels of mRNA corresponding to Sultr1;2 in these plants were no more than twice those in control plants (Figure 5). Expression of this gene was far less influenced by sulfur status than was the expression of Sutr1;1. In leaves, mRNAs corresponding to both Sultr1;1 and Sultr1;2 accumulated under severely sulfur-limited conditions (Figure 5).
Selenate affects uptake and subsequent assimilation of sulfate in plants. Previously, we found that addition of this toxic analogue in sulfur-replete medium can mimic sulfur limitation stress in plants (Takahashi et al., 2000). Sulfate pool in roots significantly decreased when treated with selenate, whereas relatively high levels of sulfate accumulated in leaves (Figure 6). It is suggested that internal movement of sulfate may be restricted under these conditions to reduce accumulation of sulfate in roots. The mRNA levels of Sultr1;1 and Sultr2;1 increased with selenate (Takahashi et al., 2000); however, the level of Sultr1;2 mRNA was not influenced (Figure 6). The patterns of mRNA accumulation of sulfate-transporter genes were fairly consistent with their responsiveness to sulfur-limitation stress (Figures 4 and 5), indicating that plants constrained to grow in the presence of selenate may suffer from sulfur deficiency.
Antisense suppression of Sultr1;1
Transgenic plants with reduced levels of Sultr1;1 mRNA were constructed by expressing antisense mRNA. A cDNA fragment of Sultr1;1 was inserted in the antisense direction under the control of cauliflower mosaic virus 35S promoter and introduced into Arabidopsis plants by Agrobacterium-mediated in planta transformation (Clough and Bent, 1998). Three independent transgenic lines, 2-3, 4-2 and 8-4, which show low levels of Sultr1;1 mRNA, were analysed (Figure 7). The antisense plants appeared identical with the wild-type plants. Additionally, both the antisense and wild-type plants were tolerant of selenium stress for at least 2 days on GM medium containing 0.1 mm of selenate.
The antisense plants were unable to accumulate Sultr1;1 mRNA in roots even in the presence of selenate in the medium where the non-transformants accumulate maximum levels of Sultr1;1 mRNA (Figure 7a). The mRNA level of Sultr1;2 was not affected by the antisense suppression of Sultr1;1 (Figure 7a). The identity between the nucleotide sequences of the Sultr1;1 antisense construct and the corresponding region of Sultr1;2 was over 70%; however, we found none of the transformant with reduced levels of Sultr1;2 mRNA. After transferring plants to 0.1 mm selenate medium, there was an increase in the size of sulfate pools in leaves of both the antisense plants and the non-transformant (Figure 7b), indicating that this increase was not related to the induction of Sultr1;1 mRNA in roots. The amount of sulfate accumulated in root was significantly lower in the antisense plants than in the non-transformant (Figure 7c) when plants were treated with 0.1 mm selenate. The difference between the root sulfate content of the antisense and the non-transformant was statistically significant (P < 0.05) (Figure 7c). These results provide direct evidence that induction of Sultr1;1 mRNA facilitates uptake of sulfate in roots.
Completion of sequencing of the genome has enabled us to identify all members of the Sultr sulfate transporter gene family in the Arabidopsis genome (The Arabidopsis Genome Initiative, 2000). As described in our previous study, these transporters were classified into four different groups based on similarities between their protein sequences (Takahashi et al., 2000). Each transporter had distinct kinetic properties, patterns of expression and cell specificity that contributed to the specific role of each in the uptake and distribution of sulfate in the plant. The regulation of high-affinity sulfate transporters appears to account for the observed increases in sulfate uptake rates under low-sulfur conditions, as these are highly upregulated in the roots of sulfur-starved plants (Bolchi et al., 1999; Smith et al., 1995; Smith et al., 1997; Takahashi et al., 2000; Vidmar et al., 1999; Vidmar et al., 2000).
In this study we have identified a novel high-affinity sulfate transporter, Sultr1;2, that is suggested to mediate constitutive uptake of sulfate under both sulfur-replete and sulfur-deficient conditions in Arabidopsis roots. Similarities of the protein sequences between Sultr1;2 and Sultr1;1 SHST1, SHST2 and HVST1 suggest that Sultr1;2 should be classified as a member of group 1 high-affinity sulfate transporters. Expression of Sultr1;2 complemented the function of sulfate transporters SUL1 and SUL2 in yeast. Sultr1;2 was functionally comparable with Sultr1;1 as a high-affinity sulfate transporter (Figure 2). Transgenic Arabidopsis plants expressing the promoter–GFP fusion gene construct suggested that Sultr1;1 and Sultr1;2 are expressed in the same cell layers of roots. GFP expressed under the control of either the Sultr1;1 and Sultr1;2 promoters was displayed in root hairs, epidermal and cortical cells of roots (Takahashi et al., 2000) (Figure 3). Our results suggest that Sultr1;1 and Sultr1;2 transporters co-localize at the root surface and play important roles in the initial uptake of sulfate. In the mature roots of these transgenic plants, GFP was preferentially found in the cortex (Takahashi et al., 2000) (Figure 3c). Absorption of sulfate from the soil and apoplastic space outside the stele is essential for subsequent distribution of sulfate through the vascular bundle, as the Casparian strips restrict free movement of solute across the endodermis. Uptake of sulfate initially occurs in both the epidermis and cortex, but it appears that the permeability of epidermal cells to sulfate may be restricted in mature regions of the root. In Sultr1;2 promoter–GFP transgenic plants, weak fluorescence of GFP was found in the guard cells (Figure 3f). Sultr1;2 is also expressed in leaves when plants are grown continuously under low-sulfur conditions (Figure 5). Sultr1;2 may supply sulfate to guard cells under sulfur-limiting conditions, but the exact contribution is not clear.
The major difference between Sultr1;1 and Sultr1;2 was the difference in their expression in response to sulfur limitation (Figures 4–6). This difference in regulation was confirmed by experiments in which plants were deprived of sulfate for 48 h. Sultr1;1 was strongly upregulated by this treatment and transcript levels increased 10-fold (Figure 4), whereas Sultr1;2 was only upregulated two- to threefold, and this occurred during the first 6 h of sulfur deprivation (Figure 4). Sultr1;1 was strongly downregulated when the external sulfate concentration increased (Figure 5). Sultr1;2, on the other hand, was far less responsive to the external sulfate concentration and remained high even when the external medium contained 1500 µm sulfate (Figure 5). Plants grown in the presence of selenate showed patterns of mRNA accumulation of Sultr1;1 and Sultr1;2 similar to those in plants deprived of sulfur (Figure 6a). When treated with selenate, the decrease in the sulfate content of roots is likely to indicate a reduced internal sulfur status of the plants, and trigger the sulfur deficiency response of Sultr1;1. The observed increase of sulfate content in leaves under such conditions suggests an irregular control of the internal transport of sulfate which results in changing the proportion of sulfate pools between leaves and roots (Figure 6b). The analysis of Sultr1;1 antisense plants shows that this phenomenon is not related to the induction of Sultr1;1 mRNA (Figure 7a). It may be associated with the regulation of vascular tissue-localizing sulfate transporters, but the exact mechanism is not clear. The level of sulfate pool in roots decreased significantly in the antisense plants (Figure 7c), indicating that accumulation of Sultr1;1 transporter can maintain uptake of sulfate, particularly under stressed conditions.
The increased expression of Sultr1;1 and, presumably, its protein product enhances sulfate-uptake capacities during sulfur limitation. It is suggested that Sultr1;2 may make a relatively small contribution to the increase in sulfate-uptake rates during sulfur limitation, but may be responsible for sulfate uptake in less sulfur-stressed plants. Sulfate uptake by plant roots may therefore be mediated by two high-affinity sulfate transporters, the highly inducible Sultr1;1 being responsible for uptake under sulfur-limiting conditions, and the more constitutively expressed Sultr1;2 ensuring sulfate uptake into plants under both sulfur-replete and sulfur-deficient conditions. Sultr1;2 has been independently identified from the analysis of selenate resistant mutants of Arabidopsis (Shibagaki et al., 2002). Those experiments indicate that a lesion in the Sultr1;2 gene restricts the uptake of both sulfate and its toxic analogue, selenate. Furthermore, no mutations were found in the Sultr1;1 gene in their screen. These results are consistent with our hypothesis that Sultr1;2 mediates the constitutive uptake of sulfate, whereas induced expression of Sultr1;1 is important for the absorption of sulfate under low-sulfur conditions in Arabidopsis roots.
Arabidopsis thaliana ecotype Columbia was used for all experiments. Plants were grown on GM media (Valvekens et al., 1988) at 22°C under 16 h/8 h light and dark cycles. GM medium contains 1700 µm sulfate as a sulfur source. Sulfate-deficient medium was prepared by replacing sulfate salts contained in MS salts (Murashige and Skoog, 1962) with equivalent chloride salts, as described previously (Takahashi et al., 2000).
Isolation of Sultr1;2 cDNA
Molecular biological experiments were carried out according to the standard protocols (Sambrook et al., 1989). The Sultr1;2 cDNA was isolated by RT–PCR. Oligonucleotide primers Sultr1;2-FE (5′-GAGCGAATTCATGTCGTCAAGAGCTCACCC-3′) and Sultr1;2-RE (5′-GCGCGAATTCTCAGACCTCGTTGGAGAG-3′) were designed to amplify the coding sequence of Sultr1;2 according to the nucleotide sequence of BAC clone F28K19 (accession no. AC009243). Total RNA was extracted from roots of 2-week-old Arabidopsis plants grown vertically on the sulfur-deficient media containing 100 µm sulfate using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was carried out at 42°C in a 20 µl solution containing 1 µg total RNA, 0.5 µg oligo (dT) primer, 10 nmol dNTPs and 200 units Superscript II reverse transcriptase (Gibco BRL, Rockville, MD, USA). PCR was carried out on the first-strand cDNA using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA). The amplified EcoRI-ended fragment was cloned into the EcoRI site of pBluescriptII SK– (Stratagene) and fully sequenced on both strands. RACE was carried out by using 5′-Full RACE Core Set (Takara, Tokyo, Japan), 3′-Full RACE Core Set (Takara) and Ex Taq DNA polymerase (Takara) following the manufacturer's protocols.
Heterologous expression of Sultr1;2 cDNA in yeast
The EcoRI-ended fragment of Sultr1;2 cDNA described above was cloned into the EcoRI site of the yeast expression vector pYE22m (Ashikari et al., 1989). The resulting plasmid was transferred into the Saccharomyces cerevisiae mutant strain, CP154-7A (Matα, his3, leu2, ura3, ade2, trp1, sul1::LEU2, sul2::URA3) (Cherest et al., 1997) by the lithium acetate method (Gietz et al., 1992). Trp+ transformants were selected on SD minimal media (Sherman, 1991) containing 20 g l−1 glucose, 0.25 mm homocysteine and required amino acids. Complementation of the mutant was carried out on the SD medium containing 0.1 mm sulfate as a sulfur source. Uptake of 35S-labelled sulfate was measured according to the method previously described by Smith et al. (1995). The Michaelis constant (Km) was calculated by fitting the first-order kinetic equation, Y = Vmax × X/(Km + X).
Transgenic Arabidopsis expressing the Sultr1;2 promoter–GFP fusion gene construct
The fusion gene construct of Sultr1;2 promoter and GFP (Chiu et al., 1996) for plant transformation was constructed as follows. Oligonucleotide primers, Sultr1;2G-FX (5′-TAGTCTCGAGTTGGTCCTACATCCCAATATCCC-3′) and Sultr1;2G-RN (5′-GCGACCATGGCTATGTAACTCTGCAAACAGAACAGG-3′) were designed according to the nucleotide sequence of BAC clone F28K19 to amplify the 5′-region flanking 2766 bp upstream of the translational initiation site of Sultr1;2 by PCR. PCR was carried out on genomic DNA prepared from Arabidopsis leaves. The XhoI–NcoI DNA fragment was cloned in pT7Blue T-vector (Novagen, Madison, WI, USA) and fully sequenced to confirm the identity with the genome. The XhoI–NcoI fragment of Sultr1;2 and NcoI–NotI fragment of the GFP coding sequence (Chiu et al., 1996) were cloned between the XhoI and NotI sites of pBluescript II SK–. The resulting Sultr1;2–GFP fusion cassette was cut out as a XhoI–SacI fragment and placed in the position of β-glucuronidase gene in pBI101 (Clontech, Palo Alto, CA, USA). This binary plasmid harbouring the Sultr1;2–GFP fusion gene was transferred to Agrobacterium tumefaciens GV3101 (pMP90) (Koncz and Schell, 1986) by electrotransformation. Arabidopsis plants were transformed according to the floral dip method (Clough and Bent, 1998). Transgenic plants were selected on GM media (Valvekens et al., 1988) containing 50 mg l−1 kanamycin sulfate. Kanamycin-resistant T2 progenies were analysed. Root tissues embedded in 5% agar were cut into 100 µm cross-sections with a microslicer (DTK-1000, Dosaka, Kyoto, Japan). Fluorescence of GFP was observed under a fluorescent microscope (Leica, DMRA) with L5 filter that provides the excitation and emission spectra at 460–500 and 512–542 nm, respectively (Leica, Wetzlar, Germany).
Preparation of total RNA and reverse transcription was carried out as described for isolation of Sultr1;2 cDNA. First-strand cDNA that derives from 0.5, 1 or 2 ng total RNA was used for amplification of Sultr1;2 and α-tubulin (Ludwig et al., 1987). First-strand cDNA that derives from 2, 4 or 8 ng total RNA was used for amplification of Sultr1;1. PCR was carried out by ExTaq DNA polymerase (Takara) using gene-specific primers for sulfate transporters and α-tubulin as follows: 1;1-FA (5′-AGTCGTTCGTCAGAGAGTGCTAGCTCCTC-3′) and 1;1-RA (5′-TGGATGTGTTTATGTATATGAATAGACGA-3′) for Sultr1;1; 1;2-F (5′-ACGGTGGACATGTTCCGATGAAACCTTCA-3′) and 1;2-R (5′-TGCGACAAGTGTAGCTTGCCTATCACCAA-3′) for Sultr1;2; TUB-F29 (5′-CTCGAAATTAGGGTTTCTACTGAGAGAAG-3′) and TUB-R29 (5′-CCGAACGAATATTTTACAGGATTTAAACA-3′) for α-tubulin. PCR was carried out for 24 cycles where cDNAs were exponentially amplified. PCR products were separated in agarose gels and stained with SYBR green (Takara). Signals were detected and quantified using an image analyser FluorImager 595 with 530DF30 filter that provides the emission spectra at 515–545 nm (Molecular Dynamics, Sunnyvale, CA, USA).
Construction of Sultr1;1 antisense plants
Transgenic plants expressing Sultr1;1 antisense cDNA was constructed as follows. Sultr1;1 cDNA fragment that covers from the position +276 bp downstream of the transcriptional initiation site to the end of the coding sequence was cloned into the BamHI site of expression vector pHTT202 in the antisense orientation (Teeri et al., 1989). Cauliflower mosaic virus 35S promoter drives the expression of the Sultr1;1 antisense RNA. This intermediary plasmid was introduced into the Ti plasmid pGV3850 of Agrobacterium tumefaciens C58C1 (Zambryski et al., 1983) by elecrotransformation and used for transformation of Arabidopsis plants (Clough and Bent, 1998). Transformants were selected on GM media (Valvekens et al., 1988) containing 50 mg l−1 kanamycin sulfate. Three representative lines (2-3, 4-2, and 8-4) showing low levels of Sultr1;1 mRNA were used for further analysis. The amount of Sultr1;1 mRNA was quantified by RT–PCR using gene-specific oligonucleotide primers 1;1-FA and 1;1-RA, as described above. These primers were designed on the 5′- and 3′-untranslated regions, which ensures amplification of the intact mRNA transcribed from the endogenous Sultr1;1 gene. For estimation of sulfate contents, plants were ground into powder under liquid nitrogen and extracted in water. Sulfate was separated and quantified by HP30 Capillary Electrophoresis System (Agilent Technologies, Palo Alto, CA, USA).
We thank Dr Y. Surdin-Kerjan (Centre National de la Recherche Scientifique, France) for the yeast mutant strain CP154-7A; Dr Y. Tanaka (Suntory Ltd, Japan) for the yeast expression vector pYE22m; and Dr Y. Niwa (University of Shizuoka, Japan) for the GFP expression vector pTH2. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, and by CREST of JST (Japan Science and Technology). H.T. was supported by a research fellowship (6067) from the Japan Society for the Promotion of Science.