Root-to-Shoot Transport of Sulfate in Arabidopsis. Evidence for the Role of SULTR3;5 as a Component of Low-Affinity Sulfate Transport System in the Root Vasculature

Distribution of Sulfate to the Aerial Part Is Facilitated by Sulfur Limitation

Uptake and rate of root-to-shoot transport of sulfate were measured using [³⁵S]sulfate. Ten-day-old plants grown on three different concentrations of sulfate (1,500, 20, and 10 μm) were incubated for 30 min in the nutrient solution containing 1,500 μm sulfate and 37 MBq L⁻¹ [³⁵S]sulfate (Table I). The results indicated that both the total uptake of sulfate and its distribution to the aerial part were significantly increased by sulfur limitation. Sulfur-starved plants grown with 10 μm sulfate exhibited approximately 10 times higher uptake of sulfate as compared to plants grown under sulfur-sufficient conditions (1,500 μm). The shoot-to-root ratio of distribution of radioactivity was 7 times higher in sulfur-starved plants than in the control (Table I). Increased root-to-shoot distribution of sulfate by sulfur deprivation suggested increased function of transport systems facilitating transport of sulfate in the vasculature.

SULTR3;5 Is Localized in the Root Vasculature

SULTR2;1 is localized in the xylem parenchyma cells and pericycles in roots (Takahashi et al., 2000) and may facilitate root-to-shoot transport. In this study, we identified a second transporter that localizes in these specific cell types. A putative open reading frame, At5g19600 that encodes the fifth sulfate transporter isoform in group 3, was isolated by reverse transcription (RT)-PCR from root RNA and designated SULTR3;5 (accession no. AB061739). The open reading frame of SULTR3;5 encoded a polypeptide of 634 amino acids, which showed typical structural features of eukaryotic sulfate transporters. The computer program (http://aramemnon.botanik.uni-koeln.de./index.html) predicted 12 membrane-spanning regions between the cytosolic N and C termini of SULTR3;5 protein.

Tissue-specific localization of SULTR3;5 in Arabidopsis was analyzed by using a fusion construct of the SULTR3;5 promoter region and green fluorescent protein (GFP; Chiu et al.,1996). The fluorescence was predominantly localized in the root, from the apex to the basal region (Fig. 1A). GFP was specifically found in the stele (Fig. 1B). A cross-section of the root indicated that the signals are localized in the xylem parenchyma and pericycle cells (Fig. 1C). These patterns of expression in roots were almost identical to those determined for SULTR2;1 (Takahashi et al., 1997, 2000). Localization of SULTR3;5 in the plasma membrane was confirmed by particle bombardment of onion (Allium cepa) epidermal cells with a GFP-tagged SULTR3;5 fusion construct. As shown in Figure 1D, the SULTR3;5-GFP fusion protein driven by the cauliflower mosaic virus (CaMV) 35S promoter was localized on the plasma membrane. These results suggested that SULTR3;5 is a plasma membrane-localizing transporter specifically expressed in the xylem parenchyma and pericycle cells of Arabidopsis roots.

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Figure 1.

Localization of SULTR3;5 in Arabidopsis. A SULTR3;5 promoter-GFP fusion gene construct was expressed in Arabidopsis (A–C). A, Whole-mount view of the seedling (bar = 2 mm). B, Longitudinal view of the root (bar = 50 μm). C, Cross-section of the root (bar = 50 μm). D, CaMV-35S promoter SULTR3;5-GFP fusion gene construct transiently expressed in the epidermal cell of onion (bar = 50 μm).

SULTR3;5-SULTR2;1 Coexpression Provides Active Transport of Sulfate in a Yeast Mutant

The yeast sulfate transporter mutant, YSD1 (Smith et al., 1995), was utilized to determine the transport functions of SULTR2;1 and SULTR3;5 that colocalize in the same cell types in the plant vascular tissue (Fig. 1). Two different types of plasmid vectors, pYE22m (Ashikari et al., 1989) and pYES2 (Invitrogen, Carlsbad, CA), were used for heterologous expression of Arabidopsis sulfate transporters in yeast cells (Fig. 2). The expression of the transporters in these vectors was driven by the yeast glyceraldehyde-3-P dehydrogenase promoter in pYE22m and the GAL1 promoter in pYES2, respectively. Figure 2A shows the growth of yeast transformants spotted on plates containing various concentrations of sulfate. SULTR3;5 was unable to complement the lesion of sulfate uptake activity in YSD1. As shown previously (Takahashi et al., 1997, 2000), yeast cells expressing SULTR2;1 substantially restored the growth of YSD1. In addition, coexpression of SULTR2;1 and SULTR3;5 resulted in a larger increase of the growth rate as compared to the single expression with SULTR2;1 alone. An increase of the growth rates of the SULTR2;1 transformants by coexpression of SULTR3;5 was observed in both combinations of the vector constructs, suggesting that stimulation of growth is relevant to the coexpression of two transporters in nature.

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Figure 2.

Complementation of the yeast sulfate transporter mutant, YSD1. A, Yeast cells expressing SULTR2;1 and SULTR3;5 sulfate transporters in YSD1 were spotted on plates containing 0.1, 0.5, or 1.5 mm sulfate. The plasmids with SULTR2;1 and SULTR3;5 cDNA inserts in pYE22m and pYES2 vectors were transformed in YSD1, and their combinations are presented at the top. Bars indicate expression of the empty vectors. The figure shows the growth after 4 d of incubation at 30°C. B, Time course influx of sulfate in yeast expressing SULTR2;1 and SULTR3;5. The uptake was measured in 0.2 mm sulfate medium. Mean ± se (n = 3).

The uptake of sulfate was measured to determine the differences between the growth rates of yeast cells expressing SULTR2;1 and SULTR3;5 individually and in combination. The uptake of sulfate increased linearly for 60 min during a time course of incubation with 0.2 mm [³⁵S]sulfate (Fig. 2B). The uptake of sulfate by SULTR3;5 was negligible and comparable to the vector control. As reported previously (Takahashi et al., 2000), SULTR2;1 expression enabled influx of [³⁵S]sulfate to the yeast cells and was able to recover growth. When SULTR2;1 was coexpressed with SULTR3;5, the uptake rate was 3-fold higher than that of SULTR2;1 alone (Fig. 2B). The sulfate uptake activity of the SULTR3;5-SULTR2;1 coexpression system was even higher than in the case of duplicated expression of SULTR2;1 using two vectors. These results suggested that SULTR3;5 may play a specific role in transporting sulfate when coexpressed with SULTR2;1.

The kinetic properties of the sulfate transport activities of SULTR2;1 and SULTR3;5-SULTR2;1 were determined (Table II). The activity of sulfate uptake followed the Michaelis-Menten kinetics as previously reported for SULTR2;1 (Takahashi et al., 2000). The calculated K_m value for the SULTR3;5-SULTR2;1 coexpression was in the range of a low-affinity sulfate transport system, which corresponds to those calculated for SULTR2;1 (Table II). By contrast, the V_max value was approximately 3-fold higher with SULTR3;5-SULTR2;1 coexpression compared to the SULTR2;1 single expression (Table II). These results suggested that SULTR3;5 is a nonfunctional sulfate transporter by itself but may participate in amplifying the capacity of SULTR2;1, as indicated by the relative increase in the V_max value of the SULTR3;5-SULTR2;1 coexpression system.

SULTR3;5 Facilitates Root-to-Shoot Transport of Sulfate

T-DNA insertion mutants of SULTR3;5 were isolated from a T-DNA-tagged population by a PCR-based reverse genetic strategy (Krysan et al., 1999; http://www.biotech.wisc.edu/Arabidopsis/). We have identified two alleles of mutant lines, each containing a single insertion of T-DNA in the coding region of SULTR3;5. T-DNA was integrated in the seventh intron and seventh exon of SULTR3;5, respectively (Fig. 3A). Each mutant line was designated sultr3;5-1 and sultr3;5-2, respectively. SULTR3;5 mRNA was predominantly found in the root tissue and showed constant levels of expression both under sulfur-sufficient and -deficient conditions in the wild type (Fig. 3B). Accumulation of SULTR3;5 mRNA was not detected in the sultr3;5 mutant (Fig. 3B). Figure 3B shows representative results of the sultr3;5-2 mutant, which has an interruption of translation of the SULTR3;5 transporter by the integration of a T-DNA within exon 7. The sultr3:5-1 mutant also showed no accumulation of SULTR3;5 and presented similar SULTR2;1 expression patterns as in the case of sultr3;5-2 (data not shown). SULTR3;5 was coexpressed with SULTR2;1 in the same cell types; however, neither the abundance nor the sulfur response of SULTR2;1 mRNA was altered by dysfunction of SULTR3;5 (Fig. 3B). SULTR2;1 was down-regulated in the shoot and up-regulated in the root by sulfur limitation, showing exactly the same expression in the wild type and in the sultr3;5 knockout (Fig. 3B).

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Figure 3.

The sultr3;5 knockout mutants. A, Structure of the SULTR3;5 genomic region and T-DNA insertion sites (arrowheads) in sultr3;5-1 and sultr3;5-2. The positions of the primers used for RT-PCR of SULTR3;5 are indicated (3;5-F1 and 3;5-R5). B, RT-PCR analysis of SULTR2;1, SULTR3;5, and α-tubulin in the wild type (Wassilewskija) and in the sultr3;5-2 mutant. Plants were grown on agar medium containing 1,500, 20, or 10 μm of sulfate for 10 d.

Figure 4 shows the rate of root-to-shoot translocation of [³⁵S]sulfate in the sultr3;5 mutants and in the wild type. Ten-day-old plants grown on three different concentrations of sulfate (1,500, 20, and 10 μm) were incubated for 30 min with 1,500 μm sulfate containing the [³⁵S] radioisotope. Two independent alleles, sultr3;5-1 and sultr3;5-2, showed a significant decrease in the shoot [³⁵S]sulfate contents when plants were pregrown on 10 μm sulfate media (Fig. 4A). By contrast, the root [³⁵S]sulfate contents of the mutants were increased compared to the wild type (Fig. 4B). Consequently, the rate of translocation calculated as a shoot-to-root ratio of distribution of [³⁵S]sulfate in the seedlings was decreased in the sultr3;5 knockouts (Fig. 4C). Significant differences between the wild type and the mutants were observed in plants pregrown under low-sulfur conditions, which allows the full induction of SULTR2;1 expression in the roots (Fig. 3B).

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Figure 4.

Distribution of sulfate in the sultr3;5 mutants. Ten-day-old plants grown on agar medium containing 1,500, 20, or 10 μm of sulfate were incubated for 30 min in the medium containing 1,500 μm sulfate and 37 MBq L⁻¹ [³⁵S]sulfate. The absolute values of sulfate transported to the shoots (A), those remaining in the roots (B), and their ratios (shoot to root; C) are indicated. Black bars and dotted bars indicate the wild type (Wassilewskija) and the sultr3;5 mutants, respectively. Statistically significant differences from the wild type are shown as asterisks (*, P < 0.05; **, P < 0.01). Mean ± se (n = 6).

Plant Materials and Growth Conditions

T-DNA insertion lines for SULTR3;5 (Wisconsin T-DNA insertion lines, Wassilewskija accession; http://www.biotech.wisc.edu/Arabidopsis/) were screened by PCR using specific primers for T-DNA and SULTR3;5. The oligonucleotide primers for SULTR3;5 are as follows: 3;5-F1 (5′-ATGGAGAATACTATAACGAGCTCTACCTC-3′) and 3;5-R (5′-CATAGCCACGAAGCAATCATAGTGGTTTC-3′). Surface-sterilized seeds of Arabidopsis (Arabidopsis thaliana) were germinated on agar medium (Fujiwara et al., 1992) and grown at 22°C under 16-h-light/8-h-dark cycles. The light intensity was 40 μmol photons m⁻² s⁻¹. The medium containing 1,500 μm sulfate represents the control condition with a sufficient supply of sulfur. A sulfate-deficient medium was prepared by replacing sulfate salts with equivalent chloride salts. Agar was rinsed three times in deionized water to remove contamination of sulfate. Ten-day-old plants were used for the analysis.

Cloning of SULTR3;5 cDNA

Molecular biological experiments were carried out according to the standard protocols (Sambrook et al., 1989). The SULTR3;5 cDNA (accession no. AB061739) was isolated by RT-PCR, and RACE primers were designed according to the nucleotide sequence of the Arabidopsis genome (The Arabidopsis Genome Initiative, 2000) presented in The Institute for Genomic Research (http://www.tigr.org/tdb/e2kl/ath1/) and Munich Information Center for Protein Sequences (http://mips.gsf.de/proj/thal/db/index.html) databases. RACE was carried out by using 5′-Full RACE Core Set (Takara, Tokyo), 3′-Full RACE Core Set (Takara), and ExTaq polymerase (Takara) following the manufacturer's protocols. Total RNA was extracted from the roots of 10-d-old Arabidopsis using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Reverse transcription and PCR were carried out using Omniscript reverse transcriptase (Qiagen) and Pfu Turbo DNA polymerase (Strategene, La Jolla, CA). The amplified PCR product was cloned into pCR-BluntII-TOPO (Invitrogen, Carlsbad, CA) and sequenced.

Generation of GFP-Fusion Transgenic Plants

The fusion gene constructs of SULTR3;5 promoter-GFP for plant transformation were constructed as follows. Oligonucleotide primers 3;5-GF1 (5′-GTCGACAACGTGGAAGAATCTGTAGCTCTTGG-3′) and 3;5-R4 (5′-ACTAGTAGAGCTCGTTATAGTATTCTCCAT-3′) were designed to amplify between the positions of −2,321 and +36 from the translation initiation site. The SpeI site at the position +36 bp was used to create translational fusion with GFP. PCR was carried out on genomic DNA prepared from Arabidopsis ecotype Columbia using Pfu Turbo DNA polymerase (Stratagene). The amplified fragment was cloned into pCR-BluntII-TOPO (Invitrogen) and sequenced. The SULTR3;5 region was cut out as a SalI-SpeI fragment and ligated into the SalI-XbaI site of the pBI101-derived promoterless GFP binary vector (Yoshimoto et al., 2003). The binary plasmids were transformed to Agrobacterium tumefaciens GV3101 (pMP90; Koncz and Schell, 1986) by the freeze-thaw method (Höfgen and Willmitzer, 1988). Arabidopsis plants were transformed according to the floral dip method (Clough and Bent, 1998). Transgenic plants were selected on GM medium containing 50 mg L⁻¹ kanamycin sulfate (Valvekens et al., 1988). Fluorescence of GFP in transgenic plants was observed under a BX61 microscope equipped with a FV500 confocal laser scanning system and a 505- to 525-nm band-pass filter (Olympus, Tokyo), as described previously (Yoshimoto et al., 2003).

Subcellular localization was studied by introducing the fusion gene construct of the SULTR3;5 coding region-GFP in onion (Allium cepa) epidermal cells. For construction of CaMV-35S promoter SULTR3;5-GFP, oligonucleotide primers 3;5F-Sal (5′-GTCGACTATGGAGAATACTATAACGAGCTCTAC-3′) and 3;5R-Nco (5′-CCATGGACACTTCCGGCTTGGTGGTGGTAAGAT-3′) were designed for PCR. The SULTR3;5 coding region was amplified by RT-PCR from root RNA. The amplified fragment was cloned into pCR-BluntII-TOPO and fully sequenced. SULTR3;5 cDNA was cut out as a SalI-NcoI fragment and cloned into the 35S-omega-sGFP (S65T; Chiu et al., 1996). Transient expression was carried out using the Biolistic PDS-1000/He particle delivery system (Bio-Rad Laboratories, Hercules, CA). The detailed procedures of bombardment are described in the operating manual based on the method of Sanford et al. (1993). Fluorescence of GFP was observed under a confocal microscope, as described above.

RT-PCR

Preparation of total RNA and reverse transcription were carried out as described in the section on cDNA cloning. First-strand cDNA that derives from 10 ng of total RNA was used for each PCR amplification. PCR was carried out by ExTaq DNA polymerase (Takara) using gene-specific primers for sulfate transporters and α-tubulin (Ludwig et al., 1987), as described previously (Yoshimoto et al., 2002). Primer sequences are as follows: 3;5-F1 (5′-ATGGAGAATACTATAACGAGCTCTACCTC-3′) and 3;5-R5 (5′-GCATGCTTGAACTGCATCGTCAATCGATA-3′) for SULTR3;5; 2;1FA (5′-TCTTCATAGTTAAACTTCCACACAACGTC-3′) and 2;1RA (5′-ACATGCAATAACCCGTAACACAACTGGTC-3′) for SULTR2;1; and 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 analyzer (FluorImager 595) with a band-pass filter that provides the emission spectra at 515 to 545 nm (Molecular Dynamics, Sunnyvale, CA).

[³⁵S]Sulfate Transport Assay in Arabidopsis

Experiments were carried out according to the method reported by Maruyama-Nakashita et al. (2004), with minor modifications. Plants were germinated on nylon mesh and grown for 10 d on vertical agar plates containing 10, 20, or 1,500 μm sulfate. Roots were submerged in the [³⁵S] radioactive liquid medium by holding the nylon mesh on a plastic frame. The labeling medium contained 1,500 μm sulfate and 37 MBq L⁻¹ [³⁵S]sulfate. After 30 min of labeling, the roots were rinsed with nonradioactive medium and then the plants were excised into roots and shoots. They were weighed separately and submerged in 500 μL of 0.1 m HCl for 1 h in the scintillation vials. The incorporated radioactivity was measured with a liquid scintillation counter (Aloka, Tokyo) after addition of 2 mL of Ultima Gold scintillation cocktail (Perkin-Elmer, Boston).

Expression of SULTR3;5 and SULTR2;1 in the Yeast Mutant

The coding region of SULTR3;5 cDNA was amplified by PCR using oligonucleotide primers 3;5-F1 (5′-ATGGAGAATACTATAACGAGCTCTACCTC-3′) and 3;5-R2 (5′-CTCGAGAAGAAGGTCACACTTCCGGCTTGG-3′). The amplified fragment was cloned into pCR-BluntII-TOPO (Invitrogen) and sequenced. The SULTR3;5 cDNA was cut out as a EcoRI-XhoI fragment and was ligated into the EcoRI-SalI site of pYE22m (Ashikari et al., 1989), or in the EcoRI-XhoI site of pYES2 (Invitrogen). The coding region of SULTR2;1 was amplified using nucleotide primers as follows: 2;1-F2 (5′-ATGAAAGAGAGAGATTCAGAGAGTTTTGA-3′) and 2;1-R (5′-TCTAGATTAAACTTTTAATCCAAAGCAAGC-3′). The amplified fragment was cloned into pCR-BluntII-TOPO and sequenced. The SULTR2;1 cDNA was cut out as a EcoRI-XbaI fragment and cloned into the EcoRI-XbaI site of pYES2. SULTR2;1 cloned in pYE22m (Takahashi et al., 2000) was also used for construction of a coexpression system. The resulting plasmids were transformed into a yeast (Saccharomyces cerevisiae) sulfate transporter-deficient mutant YSD1 by the lithium acetate method (Gietz et al., 1992), and the transformants were selected on SD medium without containing uracil and/or Trp (BD Biosciences, CLONTECH, Palo Alto, CA).

The yeast transformants were grown in SD liquid medium without sulfate salts but containing 0.25 mm homocystein thiolactone as a sulfur nutrient source (Smith et al., 1995). Gal (30 g L⁻¹) was applied to the media as a sole carbon source to activate GAL1 promoter on pYES2. The precultured yeast suspension was washed twice with the medium without sulfur source, and the cell density was adjusted to OD₆₀₀ at 1.0. The cell suspension was spotted on solid media containing a range of sulfate concentrations to determine complementation of YSD1. [³⁵S]Sulfate uptake was measured by a method that has been reported by Smith et al. (1995), with minor modifications. The uptake was terminated by centrifuging (11,000g, 1 min) 100 μL of the yeast cell suspension through 150 μL of 1:1 silicone oil (SH-550):dinonylphthalate into 14 μL of 60% perchloric acid (Nakalai Tesque, Kyoto) placed at the bottom of 250-μL microcentrifuge tubes. After centrifugation, the tips of these tubes were cut off into 2 mL of scintillant to estimate the radioactivity.

Distribution of Materials

Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining permissions will be the responsibility of the requestor.

Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank data libraries under accession numbers AB061739 (SULTR3;5 cDNA) and AB003591 (SULTR2;1 cDNA).