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Published online 3 April 2006
Published in J Environ Qual 35:726-733 (2006)
DOI: 10.2134/jeq2005.0119
© 2006 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
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TECHNICAL REPORTS

Bioremediation and Biodegradation

Constitutive Expression of a High-Affinity Sulfate Transporter in Indian Mustard Affects Metal Tolerance and Accumulation

Stormy Dawn Lindbloma, Salah Abdel-Ghanya, Brady R. Hansona, Seongbin Hwangb, Norman Terryc and Elizabeth A. H. Pilon-Smitsa,*

a Biology Department, Colorado State University, Anatomy/Zoology Building, Fort Collins, CO 80523
b Department of Molecular Biology, Sejong University, Kwangjin-Gu Kunja-Dong 98, Seoul 143-747, Korea
c Department of Plant and Microbial Biology, University of California at Berkeley, 111 Koshland Hall, Berkeley, CA 94720

* Corresponding author (epsmits{at}lamar.colostate.edu)

Received for publication April 7, 2005.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Stylosanthes hamata SHST1 gene encodes a high-affinity sulfate transporter located in the plasma membrane. In this study the S. hamata SHST1 gene was constitutively expressed in Indian mustard [Brassica juncea (L.) Czern.] to investigate its importance for tolerance and accumulation of various oxyanions that may be transported by SHST1 and for cadmium, which is detoxified by sulfur-rich compounds. The transgenic SHST1 lines SHST1-12C and SHST1-4C were compared with wild-type Indian mustard for tolerance and accumulation of arsenate, chromate, tungstate, vanadate, and cadmium. As seedlings the SHST1 plants accumulated significantly more Cd and W, and somewhat more Cr and V. The SHST1 seedlings were less tolerant to Cd, Mo, and V compared to wild-type plants. Mature SHST1 plants were less tolerant than wild-type plants to Cd and Cr. SHST1 plants accumulated significantly more Cd, Cr, and W in their roots than wild-type plants. In their shoots they accumulated significantly more Cr and somewhat more V and W. Shoot Cd accumulation was significantly lower than in wild-type, and As levels were somewhat reduced. Compared to wild-type plants, sulfur accumulation was enhanced in roots of SHST1 plants but not in shoots. Together these results suggest that SHST1 can facilitate uptake of other oxyanions in addition to sulfate and that SHST1 mediates uptake in roots rather than root-to-shoot translocation. Since SHST1 overexpression led to enhanced accumulation of Cr, Cd, V, and W, this approach shows some potential for phytoremediation, especially if it could be combined with the expression of a gene that confers enhanced metal translocation or tolerance.

Abbreviations: GSH, glutathione • PC, phytochelatin • WT, wild-type


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TOXIC METALS and metalloids are increasingly released into the environment by human activities such as industry, mining operations, use of ammunition, traffic, and agriculture, resulting in contamination that threatens natural ecosystems and human well-being (Lantzy and Mackenzie, 1979; Nriagu, 1979; Ross, 1994). A relatively new technology for environmental cleanup is phytoremediation, which uses plants and their associated microbes to extract, degrade, or stabilize pollutants. Metal extraction into harvestable plant tissues may be further enhanced by genetic engineering. Already, transgenic plants with enhanced metal tolerance and accumulation have been created via (over)expression of metal transporter proteins (Samuelsen et al., 1998; Arazi et al., 1999; Van der Zaal et al., 1999; Curie et al., 2000; Hirschi et al., 2000). The purpose of this study was to test the role of the sulfate transporter in tolerance to and accumulation of various metal(loid)s.

Sulfur is an essential element for plant primary metabolism as a structural component of proteins and lipids, antioxidants, regulatory molecules, metal-binding molecules, and cofactors and coenzymes. Plants take up sulfur primarily in the form of sulfate. After uptake most sulfate is incorporated into organic molecules via the sulfate assimilation pathway. It is activated by ATP sulfurylase to form adenosine phosphosulfate, followed by a reduction by APS reductase to form free sulfite, which is coupled to O-acetylserine to form cysteine (Setya et al., 1996). Cysteine can be incorporated into proteins, further metabolized to methionine and its derivatives, or used for the production of the antioxidant glutathione (GSH), and the metal-binding peptides phytochelatins (PCs). Under conditions of oxidative stress such as the presence of heavy metals there is an increased demand for reduced S compounds like GSH and PCs, and genes involved in uptake and reduction of sulfate are upregulated at the transcription level under these conditions (Leustek et al., 2000; Nocito et al., 2002), as are genes involved in formation of GSH and PCs (Xiang and Oliver, 1998).

The transport of sulfate over plant membranes is mediated by sulfate transporters. There are many different sulfate transporters in plants that differ in intracellular location, expression pattern, and kinetic properties. In Arabidopsis 14 sulfate transporters have been reported that can be divided into five distinct groups with different kinetic properties (Hawkesford, 2003). Sulfate can enter plants via a Group 1 high-affinity sulfate transporter in the plasma membrane (Smith et al., 1995; Shibagaki et al., 2002; Yoshimoto et al., 2002). Group 1 high-affinity sulfate transporters are expressed mainly in plant roots, and are up-regulated under sulfur starved conditions (Yoshimoto et al., 2002).

There is evidence that sulfate transporters can also mediate the transport of related oxyanions: all sulfate transporters tested can also transport selenate (Smith et al., 1995; Hawkesford, 2003), and sulfate transport is inhibited by selenate, arsenate, chromate, molybdate, and tungstate (Wilson and Bandurski, 1958; Leustek, 1996). If sulfate transporters indeed transport other oxyanions, their constitutive overexpression may lead to enhanced uptake of these elements, a property that would be desirable for phytoremediation. In addition, it is feasible that constitutive expression of sulfate transporters leads to enhanced production of S-rich metal binding peptides (GSH, PCs), which may enhance metal tolerance and accumulation.

To investigate the effect of overexpression of a sulfate transporter on metal tolerance and accumulation, Indian mustard plants were engineered to constitutively express the Stylosanthes hamata SHST1 gene (Smith et al., 1995), encoding a high-affinity sulfate transporter that is thought to be involved in root sulfate uptake over the cell membrane. The resulting SHST1 transgenic plant lines were compared to wild-type (WT) Indian mustard with respect to tolerance and accumulation of As, Cd, Cr, Mo, V, and W, supplied individually to seedlings or mature plants.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material
Indian mustard wild-type seeds were from accession no. 173874, North Central Regional Plant Introduction Station (Ames, IA). Transgenic SHST1 plants were obtained via transformation of plants from this same accession number with a DNA construct containing the Stylosanthes hamata SHST1 cDNA sequence (kind gift from Dr. F.W. Smith, CSIRO, Australia) under the 35S CaMV constitutive promoter in binary vector pJD301 (kind gift from Dr. V. Walbot, Stanford University, Palo Alto, CA). The kanamycin-resistance gene (nptII) under control of the nopaline synthase promoter was used as a marker gene. Hypocotyls of Indian mustard were transformed using Agrobacterium tumefaciens–mediated transformation as described in Pilon-Smits et al. (1999). Seven independent transgenic lines were obtained; two of them, SHST1-4C and SHST1-12C, were selected for further studies. For all of the studies homozygous T3 plants were used.

Elemental Forms and Concentrations
The chemical forms and concentrations of the metal(loid)s used for the seedling treatments were 25 mg L–1 arsenic(V) as Na2HAsO4, 10 mg L–1 cadmium as CdSO4, 5 mg L–1 chromium as K2CrO4, 20 mg L–1 vanadium as Na3VO4, 85 mg L–1 molybdenum as (NH4)6Mo7O24, and 100 mg L–1 tungsten as Na2WO4. The same chemical forms of metal(loid) were supplied to mature plants in a hydroponic system at 16 mg L–1 As, 5.6 mg L–1 Cd, 6 mg L–1 Cr, 6 mg L–1 V, and 50 mg L–1 W. No precipitation was observed visually when these metal salts were added to agar medium or nutrient solution.

Northern Blotting
Expression of the S. hamata SHST1 gene in the two SHST1 plant lines was analyzed at the mRNA level essentially as described by van Huysen et al. (2003). Total RNA from 3-wk-old wild-type and transgenic (SHST1-4C and SHST1-12C) Indian mustard plants was isolated using the TRIzol reagent method (Invitrogen, Carlsbad, CA). Ten micrograms of total RNA was separated on a 1% (w/v) agarose gel containing 4% formaldehyde, and transferred to a nylon membrane (Hybond N; Amersham, Little Chalfont, UK). The RNA was cross-linked to the filter by exposing it to UV light in a Stratalinker (Stratagene, La Jolla, CA) and probed with a 33P-labeled 1.0 kb SHST1 cDNA obtained using the SHST1-F (5'-CCACCTAAGCAGACACTCTTCC-3') and SHST1-R (5'-CATGGAAGAAGATTTCATTAGC-3') primers. Radioactive probe was synthesized with a DECAprime II labeling kit from Ambion (Austin, TX) using nanonucleotide random primers. Hybridization was performed at 42°C in a solution containing 50% formamide, 5X SSPE, 5X Denhardt's solution, 0.1% (w/v) SDS, and 100 µL mL–1 salmon sperm DNA. After hybridization, the membrane was washed with 0.1 X SSC and 0.1% SDS at 65°C, and radioactive bands were visualized and quantified in a PhosphorImager (STORM; Molecular Dynamics, Sunnyvale, CA). As a control for loading, the 28S ribosomal RNA band was quantified from the ethidium bromide-stained gel, using Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD). The intensities of the SHST1 bands were quantified using ImageQuant (Molecular Dynamics, Sunnyvale, CA). The relative expression levels of SHST1 were then calculated as a ratio of the detected SHST1 bands to the amount of 28S ribosomal RNA.

Metal Tolerance and Accumulation Experiments
Seedlings
To compare SHST1 transgenic plant lines to WT Indian mustard plants for metal tolerance at the seedling level, seeds of each plant line (SHST1-4C, SHST1-12C, and WT) were surface-sterilized as described by Pilon-Smits et al. (1999) and grown on agar medium containing one of the selected metals. The seeds were sown in Magenta tissue culture boxes (Sigma, St. Louis, MO). Each experiment was performed in parallel with its own control (i.e., using growth medium lacking the additional metal or metalloid). The agar medium contained half-strength Murashige and Skoog (MS) salts and vitamins (Murashige and Skoog, 1962), 10 g L–1 sucrose, and 4 g L–1 agargel (Sigma); individual metals were added to the final concentrations as described earlier in this section. These concentrations were aimed to give an approximately 50% reduction in wild-type seedling fresh weight, corresponding with an approximately 75% reduction in seedling root length compared to untreated controls. The Magenta boxes with the different treatments and the control boxes were randomly arranged and the seedlings were allowed to grow for 7 d in a growth chamber at 25°C and a 16-h photoperiod. Individual seedlings were then harvested and washed, and root length was measured as a parameter for metal tolerance (Murphy and Taiz, 1995). To correct for any differences between experiments, metal tolerance was expressed as relative root length, calculated as root length in the presence of the metal(loid) divided by root length on control medium. The plant material was then dried and prepared for elemental analysis.

Mature Plants
To compare metal tolerance and accumulation at the mature plant level, seeds of WT and SHST1 transgenics were surface-sterilized, sown on agar medium as described above, and grown for 4 d. The seedlings were then transferred to sand, and watered with half-strength Hoagland's nutrient solution daily (Hoagland and Arnon, 1938) in a greenhouse at 25°C, 16-h photoperiod until they were 5 wk old. The plants were then transferred to a greenhouse nutrient film technique setup as described by Zhu et al. (1999a). After 1 wk of adjustment to the new medium, the fresh weights of the plants were measured and the individual metal treatments were started. One metal was supplied at a time in the form and concentration described earlier in this section; for each experiment a control treatment without the metal(loid) was run in parallel. These metal concentrations were chosen so as to give an approximately 50% reduction in fresh weight in mature plants compared to untreated controls. Ten replicate plants were used per plant line (WT, SHST1-4C, or SHST1-12C) per treatment. The nutrient solutions were replaced every 3 d for a total of 14 d, after which the plants were harvested, washed, weighed, and dried for elemental analysis.

Elemental Analysis
Plant tissues were dried for 48 h at 70°C for elemental analysis. Five dried shoot samples of seven seedlings per treatment were weighed and acid-digested according to the method of Zarcinas et al. (1987). The total elemental concentrations in the digests were measured using inductively coupled plasma atomic emission spectrometry (ICP–AES; Thermo Jarrell Ash, Franklin, MA) according to the method of Fassel (1978), using appropriate standards and quality controls. Individual shoots and roots of mature plants were ground in a Wiley mill, acid-digested, and analyzed by ICP–AES as described above.

Statistical Analyses
The software program JMP-IN (SAS Institute, 2005) was used for statistical analysis of metal tolerance and accumulation data. Analysis of variance (ANOVA) was performed followed by pairwise post-hoc analyses to determine which means differed significantly ({alpha} = 0.05). Statistically significant differences (P < 0.05) are reported in the text and shown in the figures and tables.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Creation of SHST1 Transgenics
When Indian mustard plants were transformed with a DNA construct containing the Stylosanthes hamata SHST1 gene under the 35S CaMV constitutive promoter, seven independent transgenic lines were obtained. Two of them, SHST1-4C and SHST1-12C, were selected for further studies. The two lines chosen express the S. hamata SHST1 gene to different levels judging from Northern blotting (Fig. 1): SHST1-12C shows a higher SHST1 expression than SHST1-4C. No mRNA was detected in wild-type Indian mustard using the S. hamata SHST1 probe, suggesting that none of the endogenous sulfate transporters in Indian mustard was homologous enough to the SHST1 gene to be detected. Judged from segregation analysis and Southern blotting both lines had one insertion of the transgene (results not shown).


Figure 1
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  		Fig. 1. Transcript levels of S. hamata SHST1 in transgenic Indian mustard lines. Total RNA was isolated from 3-wk-old wild-type (WT) and SHST1 overexpressing (SHST1-4C and SHST1-12C) Indian mustard plants. (A) Ethidium bromide-stained gel showing total RNA loading. (B) Autoradiogram of RNA blot after hybridization with 33P-labeled SHST1 cDNA.

 
Metal Tolerance and Accumulation at the Seedling Level
The transgenic SHST1 lines were first compared with WT Indian mustard with respect to metal tolerance and accumulation in seedlings (Fig. 2). The metals tested were As, Cd, Cr, Mo, V, and W. These metals were chosen because most occur as oxyanions (negative ions containing oxygen) as does sulfate. Cadmium was included because this cation is known to be bound by S ligands in plants (Pickering et al., 2000). The forms and concentrations of the metal(loid)s used are described in the Materials and Methods section.


Figure 2
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  		Fig. 2. Seedling metal tolerance for wild-type (WT) and SHST1 overexpressing (SHST1-4C and SHST1-12C) Indian mustard plants. Shown is the ratio of seedling root length grown on medium containing metal(loid) relative to root length on control medium. The form and concentration used for each metal(loid) is described in Materials and Methods. Shown are means ± SE (n = 36). The letters above the bars indicate statistically significant differences between groups (ANOVA with pairwise post-hoc analyses, {alpha} = 0.05).

 
Both of the SHST1 transgenic plant lines were less tolerant than WT to cadmium, molybdate, and vanadate. There were no differences in tolerance between the plant lines when grown on media containing chromate or tungstate. The two SHST1 plant lines gave inconsistent results for As, one line being more tolerant, and one being less tolerant than WT seedlings.

The SHST1 transgenic lines both contained higher W levels in their shoots than WT seedlings (Fig. 3). The shoot levels of Cr and V were also higher in SHST1 lines than in WT, but there were no significant differences among the plant lines for these elements. SHST1-4C, the least arsenate-tolerant plant line, accumulated more As than SHST1-12C or WT seedlings. The SHST1-12C plant line accumulated more Cd than SHST1-4C or WT seedlings. There were no differences in accumulation for Mo. Incidentally, the drying temperature used may have led to some loss of tissue As, so the actual As levels may have been somewhat higher.


Figure 3
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  		Fig. 3. Seedling metal accumulation for wild-type (WT) and SHST1 overexpressing (SHST1-4C and SHST1-12C) Indian mustard plants. Shown is the shoot metal concentration after 7 d of growth on agar medium supplied with a metal(loid) to a concentration as indicated in Materials and Methods. Shown are means ± SE (n = 5 samples pooled from 14 plants each). The letters above the bars indicate statistically significant differences between groups (ANOVA with pairwise post-hoc analyses, {alpha} = 0.05).

 
Supplying the seedlings with toxic levels of a metal(loid) affected the plant's ability to accumulate essential elements. Interactions between supplied metal(loid) and the essential elements Cu, Fe, Mg, S, and Zn were investigated, as were differences between control and treated plants for each plant line. Only the results for sulfur are shown in Table 1; significant differences for the other elements are summarized in the text. The Fe levels were higher in SHST1 seedlings than in WT when grown on Cd, vanadate, or tungstate. SHST1 seedlings accumulated more Mg than WT after As or Cd treatment, but less Mg than WT after V or W treatment. Sulfur concentration was higher in SHST1 seedlings than in WT under Cd or Cr treatment, but lower than WT under V or W treatment. Zinc levels were higher in SHST1 plants than in WT when treated with As, Cd, and W. Incidentally, in the seeds used the S levels were slightly (5–10%) lower in the SHST1 transgenics compared to wild-type seeds, but not significantly so (P = 0.10 ANOVA). Therefore the differences in tissue S levels are likely due to uptake differences.


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  		Table 1. Shoot concentrations of S in wild-type (WT) and SHST1 overexpressing (SHST1-4C and SHST1-12C) Indian mustard seedlings after treatment with various metal(loid)s. Values shown represent the average and standard error of five replicate samples, collected from eight seedlings each.

 
The effects of the metal treatments on plant nutrient levels varied between SHST1 and WT plants in some respects. When treated with Cd or Mo, the Cu levels in WT seedlings were higher than in WT grown under control conditions. In contrast, these treatments did not significantly affect Cu accumulation in the SHST1 seedlings. The tungstate treatment reduced Fe accumulation in WT plants, while it enhanced Fe accumulation in SHST1 plants. Magnesium accumulation was reduced in WT seedlings by the Cd treatment, while in SHST1 seedlings it was not. Sulfur accumulation was enhanced by the Cd treatment in SHST1 seedlings, but not significantly in WT seedlings (Table 1). SHST1 seedlings treated with arsenate had higher Zn levels in their shoots compared to their own controls, whereas WT seedlings had reduced shoot Zn levels. SHST1 seedlings treated with molybdate had reduced Zn accumulation compared to control SHST1 seedlings, while the Mo treatment did not significantly affect Zn accumulation in WT seedlings.

Metal Tolerance and Accumulation at the Mature Plant Level
Metal Tolerance
To test the role of the SHST1 sulfate transporter in metal(loid) tolerance at the mature plant level, the two SHST1 transgenic plant lines were compared to wild-type Indian mustard in a hydroponic setup containing the metal(loid) of interest (Fig. 4). The form and concentration of the metal(loid) used is described in the Materials and Methods section. Molybdate was not included in the mature plant study since there were no differences in Mo accumulation at the seedling level. Metal tolerance was calculated for each plant line as plant growth in the presence of the metal as a fraction of its growth under control conditions. The SHST1-12C plants were less tolerant than WT when supplied with cadmium or chromate; the SHST1-4C line was intermediate in this respect. There were no differences in tolerance between the plant lines when grown on media containing arsenate, vanadate, or tungstate.


Figure 4
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  		Fig. 4. Mature plant metal tolerance for wild-type (WT) and SHST1 overexpressing (SHST1-4C and SHST1-12C) Indian mustard plants. Shown is the ratio of plant fresh weight when grown on medium containing metal(loid) relative to its fresh weight on control medium. The form and concentration used for each metal(loid) is described in Materials and Methods. Shown are means ± SE (n = 10). The letters above the bars indicate statistically significant differences between groups (ANOVA with pairwise post-hoc analyses, {alpha} = 0.05).

 
Shoot Accumulation of Metals and Their Effects on Essential Nutrient Levels
There were differences in shoot accumulation of Cd and Cr between the SHST1-4C, SHST1-12C, and WT plant lines (Fig. 5). The SHST1-12C plants contained less Cd in their shoots when treated with Cd, and more Cr when treated with Cr compared to WT plants. No differences were found between the plant lines with respect to shoot As, V, or W accumulation, although the V and W levels in the shoots of SHST1 plants were somewhat higher, and As levels lower compared to WT plants.


Figure 5
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  		Fig. 5. Mature plant shoot metal accumulation for wild-type (WT) and SHST1 overexpressing (SHST1-4C and SHST1-12C) Indian mustard plants. Shown is the shoot metal concentration after hydroponically grown plants were treated for 14 d with a metal(loid) concentration as indicated in Materials and Methods. Shown are means ± SE (n = 10). The letters above the bars indicate statistically significant differences between groups (ANOVA with pairwise post-hoc analyses, {alpha} = 0.05).

 
Interactions in mature plant shoots were investigated between supplied metal(loid) and the essential elements Cu, Fe, Mg, S, and Zn. Again, only the results for sulfur are shown (Table 2). SHST1 plants grown in solution with Cd or W had less Fe in their shoots than WT plants. Shoots of SHST1-12C plants contained less S than WT plants after treatment with Cd or vanadate; SHST1-4C plants were intermediate in this respect (Table 2). While the Cd treatment significantly reduced Mg and S levels in the shoots of the SHST1 plants compared to control conditions, it did not in WT plants. Also, vanadate significantly reduced shoot Zn levels in SHST1 plants but not in WT plants.


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  		Table 2. Shoot and root concentrations of S (mg kg–1 dry weight) in mature wild-type (WT) and SHST1 overexpressing (SHST1-4C and SHST1-12C) Indian mustard plants after treatment with various metal(loid)s. Values shown represent the average and standard error of 10 replicate plants, each sampled once.

 
Root Accumulation of Metals and Effects on Essential Nutrient Levels
The roots of the SHST1 plants contained higher Cr levels than WT roots (Fig. 6). SHST1-4C plants also accumulated Cd and W to higher concentrations than WT plants, and the SHST1-12C plants were intermediate in this respect. As and V root accumulation was not significantly different between the plant lines.


Figure 6
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  		Fig. 6. Mature plant root metal accumulation for wild-type (WT) and SHST1 overexpressing (SHST1-4C and SHST1-12C) Indian mustard plants. Shown is the root metal concentration after hydroponically grown plants were treated for 14 d with a metal(loid) concentration as indicated in Materials and Methods. Shown are means ± SE (n = 5 samples pooled from two plants each). The letters above the bars indicate statistically significant differences between groups (ANOVA with pairwise post-hoc analyses, {alpha} = 0.05)

 
Root concentrations of the essential elements Cu, Fe, Mg, S, and Zn were also determined. The results for sulfur are shown in Table 2; results from the other metals are summarized in the text. There were higher levels of Mg in the roots of SHST1 plants compared to WT roots after all of the treatments. Moreover, SHST1 plants contained more S in their roots than WT plants after treatment with As, Cd, Cr, or V. Root Fe levels in SHST1 plants were lower than in WT plants when treated with arsenate, chromate, or vanadate. Vanadate-treated SHST1-12C plant roots also had lower root Zn levels than WT; SHST1-4C plants were intermediate. Arsenic treatment enhanced root S and Mg levels in the SHST1 plant lines compared to control conditions, but in the WT plants it did not.

Translocation
The root-to-shoot translocation factors for the supplied metal(loid)s are shown in Table 3. Of the metals tested W was translocated from the roots to the shoots to a much larger extent than the other elements: root-to-shoot ratios were around 1 for W. Arsenic was translocated roughly 10-fold less, Cr and V 20- to 40-fold less, and Cd was translocated the least. The SHST1 plants translocated Cd to a lesser degree than the WT plants, and As translocation was also somewhat lower (NS). In contrast, the SHST1-12C plants translocated more Cr and more V than the other plant lines. Sulfur translocation (Table 3) was lower in SHST1 lines treated with As, Cd, and V compared to the WT plants. Under control conditions the SHST1 plants also tended to have a lower degree of S translocation (NS) compared to WT plants.


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  		Table 3. Root-to-shoot translocation in wild-type (WT) and SHST1 overexpressing (SHST1-4C and SHST1-12C) Indian mustard plants treated with different elements, expressed as the ratio of shoot (Fig. 5) to root (Fig. 6) concentration.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The S. hamata SHST1 transporter is a Group 1 high-affinity sulfate transporter expressed in the cell membrane, primarily in root cells (Smith et al., 1995). In this study SHST1 was constitutively expressed to gain insight into the involvement and importance of this transporter for uptake and translocation of sulfate, other supplied oxyanions, and Cd. Results presented here from experiments with seedlings suggest that the SHST1 transporter can mediate the uptake of WO42– and possibly of VO43– and CrO42– since both SHST1 lines accumulated more W and to a lesser extent more Cr and V compared to the WT seedlings. Treatment with arsenate, vanadate, and Cd generally enhanced seedling S levels, as did chromate but only in the SHST1 seedlings. These enhanced S levels may have been due to upregulation of endogenous sulfate transporters. Such upregulation is triggered by S deficiency, particularly by low levels of reduced S compounds such as GSH (Lappartient et al., 1999). Lower levels of reduced S compounds after metal treatment could be explained either by competition of the supplied oxyanions with S analogs for S-metabolism enzymes, or by enhanced use of GSH for detoxification of the elements. It is known that under metal stress GSH is used to form phytochelatins, which bind As and Cd and perhaps other metals (Pickering et al., 2000). Cadmium treatment is known to lead to induced expression of sulfate transporters via its interactions with reduced S compounds (Leustek et al., 2000; Nocito et al., 2002). The higher Cd levels observed in the SHST1 seedlings compared to WT suggest that their higher S levels facilitated Cd accumulation. The higher S levels did not appear to help SHST1 seedlings detoxify the metal, since Cd tolerance was not enhanced but rather decreased in SHST1 plants, possibly due to the direct toxic effects of their higher Cd levels. Tolerance to molybdate and vanadate was also lower in SHST1 seedlings than in WT. For V this may be due to their somewhat higher shoot V levels. The lower Mo tolerance cannot be readily explained since shoot Mo levels were comparable. It is possible that seedling root Mo levels were higher; this was not analyzed for lack of sufficient plant material.

From results with mature plants it appears that the SHST1 transporter facilitates uptake of the oxyanions WO42–, CrO42–, and perhaps VO43–. Constitutive expression of SHST1 also facilitated uptake of SO42– and Cd into roots, but not their translocation to the shoot. Thus, the results from seedlings and mature plants were fairly consistent, except that the additional Cd and S taken up by SHST1 roots was not translocated to the shoot in mature plants. The lower translocation of Cd and S (as well as As) in SHST1 transgenics may be due to enhanced Cd (and As) sequestration in the roots by the S-rich thiols GSH and PCs, preventing further transport in/into the vascular tissue. It is also possible that the endodermis, which acts as a barrier for root-to-shoot transport, is still developing in 7-d-old seedlings. Once the endodermis is fully established the root-to-shoot translocation depends on export of the ions out of the root symplast into the root xylem. This process is likely not mediated by SHST1 and thus limited by the activity of endogenous transporters.

Translocation of sulfate to the shoot via the xylem is thought to be facilitated by sulfate transporters from Groups 4, 3, and 2 in Arabidopsis roots, involved in vacuolar efflux (sultr4;1 and 4;2) and xylem loading (sultr3;5 and 2;1), respectively (Takahashi et al., 1997, 2000, personal communication). Sulfate uptake from the shoot xylem into leaf mesophyll cells may involve the combined action of Group 2 and 3 sulfate transporters (Takahashi et al., 1999; Grossman and Takahashi, 2001). These same sulfur transporters may be involved in translocation of the different oxyanions used in this study. The affinity of the individual transporters for the different oxyanions likely varies. This would explain the differences in translocation between the different elements. Since plants typically contain more than 10 sulfate transporters and the endogenous sulfate transporters in Indian mustard are not well characterized at this time, it would be difficult to determine which one(s) may have been responsible for the observed effects. Tungsten differed remarkably in translocation from the other elements tested, showing a root-to-shoot ratio that was close to one. Perhaps there is no barrier limiting root-to-shoot translocation for this element (e.g., if it crosses membranes via passive transport).

The lower tolerance of the SHST1 plants to Cd may in part be due to Cd-induced nutrient deficiency. Cadmium treatment of mature plants resulted in a decrease in shoot concentration of Mg and S in SHST1 plants but not in WT. These lower nutrient levels in SP shoots are expected to impair photosynthesis and thus productivity. In addition, the higher root Cd levels in SHST1 plants may have led to a direct toxic effect (e.g., by mediating oxidative stress). The lower tolerance of the SHST1 plants to Cr is not apparent from nutrient levels and may be due to a direct toxic effect of chromate at the cellular level, since Cr levels were higher in SHST1 plants.

Does constitutive expression of a high-affinity sulfate transporter like SHST1 show any promise for breeding plants with enhanced phytoremediation capacity? The SHST1 plants showed enhanced uptake of Cr, Cd, V, and W, which would be an attractive property for use in phytoextraction, where plants are used to accumulate pollutants followed by harvesting of the plant material. The reduced tolerance of the SHST1 plants to Cd and Cr and their inability to translocate the accumulated Cd to the shoot may be overcome by concomitant overexpression of genes that enhance metal tolerance or translocation. Enhanced tolerance to Cd, for instance, may be conferred by overproduction of phytochelatins (Zhu et al., 1999a, 1999b). Simultaneous expression of two genes, one conferring enhanced uptake and one enhanced tolerance, has been used successfully for As (Dhankher et al., 2002). In addition, if nutrient deficiency appears to be a cause of the reduced tolerance, this effect may be alleviated by fertilization with the nutrients in question. Increased translocation may be achieved by simultaneous overexpression of a transporter involved in xylem loading or unloading. Certain Group 2, 3, and 4 sulfur transporters may be possible candidates, but as mentioned above much remains to be discovered about the nature of these transporters in Indian mustard.


   ACKNOWLEDGMENTS
 
We thank Dr. Frank Smith for generously providing the SHST1 cDNA, and Dr. Virginia Walbot for providing the binary vector. This work was supported by U.S. National Science Foundation Grant #MCB-9982432 awarded to Elizabeth A. H. Pilon-Smits, including a Research Experience for Undergraduates supplement for Stormy Dawn Lindblom.


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