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First published online January 5, 2007; 10.1104/pp.106.091462 Plant Physiology 143:1231-1241 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Characterization of a Selenate-Resistant Arabidopsis Mutant. Root Growth as a Potential Target for Selenate Toxicity1,[OA]Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Ecole Nationale Supérieure Agronomique de Montpellier, Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, Université Montpellier II, 34060 Montpellier, France (E.E.K., N.C., H.R., P.F., P.B., J.-C.D.); and Department of Plant and Microbial Biology, University of California, Berkeley, California 94720–3102 (N.T.)
Screening an Arabidopsis (Arabidopsis thaliana) T-DNA mutant library for selenate resistance enabled us to isolate a selenate-resistant mutant line (sel1-11). Molecular and genetic characterization showed that the mutant contained a lesion in the SULTR1;2 gene that encodes a high affinity root sulfate transporter. We showed that SULTR1;2 is the only gene among 13 mutated genes of the Arabidopsis sulfate transporter family whose mutation conferred selenate resistance to Arabidopsis. The selenate resistance phenotype of the sel1-11 mutant was mirrored by an 8-fold increase of root growth in the presence of selenate as shown by the calculated lethal concentration values. The impairment of SULTR1;2 activity in sel1-11 resulted in a reduced 35S-sulfate uptake capacity by both roots and calli and a reduced sulfate and selenate content in root, shoot, and calli. Comparing sulfate-to-selenate ratios instead of absolute sulfate and selenate contents in roots and shoots enabled us to gain better insight into the mechanism of selenate toxicity in Arabidopsis. Roots of the sel1-11 mutant line showed a higher sulfate to selenate ratio than that of wild-type roots, while there were no significant differences in sulfate to selenate ratios in shoots of wild-type and mutant lines. These results indicated that the mechanism that confers the selenate resistance phenotype to the sel1-11 line takes place rather in the roots. It might be in part the result of a lower selenate uptake and of a protective effect of sulfate against the toxic effects of selenate on root growth. These results revealed in plants a central and specific role of the transporter SULTR1;2 in selenate sensitivity; they further suggested that root growth and potentially the root tip activity might be a specific target of selenate toxicity in Arabidopsis.
In animals, both the beneficial and toxic effects of selenium are well documented (Ohlendorf et al., 1986  ; Läuchli, 1993; Spallholz, 1994; Arteel and Sies, 2001; El-Bayoumy, 2001; Tapiero et al., 2003; Schromburg et al., 2004). They are, however, poorly understood in plants and yeast (Saccharomyces cerevisiae; Läuchli, 1993; Terry et al., 2000). Although required in minute quantities (0.05–0.10 mg/kg) by animals to form the selenoenzymes, thioredoxin reductase and glutathione peroxidase, selenium quickly becomes toxic at higher concentrations (2–5 mg/kg dry food) causing mortality, developmental defects, and reproductive failure (Terry et al., 2000). Selenium has not yet been found to have any role in higher plants, although some resistant plants growing on seleniferous soils, including some species of Astragalus and Stanleya, are able to accumulate selenium to very high concentrations (Feist and Parker, 2001; Pickering et al., 2003). It has, however, been shown to be essential in Chlamydomonas reinhardtii (Novoselov et al., 2002). In most plants, protein synthesis is adversely affected, causing symptoms including chlorosis and stunting that mimic sulfate starvation, as well as withering and drying of leaves and premature death (Terry et al., 2000).
Sulfate (SO42–) is the main source of sulfur taken up by roots from the soil solution. Because of the structural similarity of selenate (SeO42–) to sulfate, selenium (as selenate) competes for access to membrane sulfate transporters and to enzymes of the sulfur assimilation pathway, leading to the reduction and assimilation of selenate to the selenium analogs of Cys and Met, seleno-Cys and seleno-Met, in plants (Leggett and Epstein, 1956
Plant responses to sulfur deficiency are relatively well documented in Arabidopsis and include the induction of SULTR1;1 expression in roots, which contributes to the increased root sulfate uptake capacity (Takahashi et al., 2000
One of the main mechanisms of selenium toxicity in animals and plants is believed to be the nonspecific incorporation of selenium into proteins (Brown and Shrift, 1981
One approach to elucidating mechanisms of selenium toxicity in plants is to use mutant lines. Screening of an ethyl methanesulfonate (EMS) mutant library of Arabidopsis led to the identification of allelic selenate-resistant mutants (sel1) that had lesions in the SULTR1;2 gene encoding a root sulfate transporter (Shibagaki et al., 2002
Isolation and Genetic Characterization of Selenate-Resistant Mutants
Selenate-resistant mutants of Arabidopsis were identified by germinating T2 seeds of a T-DNA mutagenized Wassilewskija (WS) ecotype population on a solid germinating medium (GM) containing 10 µM selenate and djenkolate as an organic sulfur source (Greenberg et al., 1964
Genetic analysis indicated that the selenate-resistant phenotypes of the mutants A25, B78, and B181 are caused by single recessive mutations (Table I
). Selenate sensitivity in the F2 progeny of the three mutant lines segregated in a 3:1 ratio (sensitive:resistant), indicating that the mutant phenotypes are caused by a recessive mutation at a single locus. None of the four mutant lines showed resistance to kanamycin (data not shown), a character normally conferred by the T-DNA insertion (Bechtold and Pelletier, 1998
Mapping and Identification of the Mutations in the SULTR1;2 Gene
To identify the gene responsible for the mutant phenotype, the lesions in A25 and B78 were genetically mapped. These two mutants were crossed with a wild-type plant from the Landsberg erecta ecotype. The F1 plants were allowed to self fertilize and the resulting F2 plants used to genetically map the mutation. F2 seeds were germinated on solid GM medium containing 10 µM selenate and scored for selenate resistance. Total DNA was isolated from F2 plants individually, and the segregation of molecular genetic markers was analyzed. The mutation was mapped to the lower arm of chromosome 1 near the marker alcohol dehydrogenase (ADH; Fig. 1A
) by examining 51 and 76 F2 progeny from A25 and B78, respectively. Nine allelic selenate-resistant mutants (sel1-1 to 9) were previously isolated by screening for plant growth on medium containing 10 µM selenate (sel1-1 to 7) or 20 µM selenate (sel1-8 and sel1-9; Shibagaki et al., 2002
We examined the nature of the mutations in the SULTR1;2 gene in the four isolated mutant lines. PCR analysis indicated that sel1-12 and sel1-14 bear an important deletion in the lower arm of chromosome 1, covering 65 kb and concerning 16 different genes, including SULTR1;2 (Fig. 1B). Sequencing of the SULTR1;2 gene of sel1-11 and sel1-13 indicated the presence of the same microdeletion of seven bases at the start of exon number 2 (Fig. 1C). Bioinformatics analysis indicated that this microdeletion generates a frame shift, the creation of a premature stop codon and subsequently the synthesis of a truncated protein of 139 amino acids instead of the usual 653 amino acids of the SULTR1;2 native protein. Subsequently, we chose the sel1-11 mutant line for further characterization. SULTR1;2 was previously characterized as a high affinity sulfate transporter (Yoshimoto et al., 2002 ; Shibagaki and Grossman, 2004). We confirmed the transport function for SULTR1;2 by complementing the yeast sulfate transport defective 1 strain (YSD1) yeast mutant defective in its sulfate transport capacity (Smith et al., 1995). The calculated kinetic parameters values, Km for sulfate (approximately 7.4 µM) and Vmax (approximately 0.6 nmol h–2 [108 cellules]–1), were consistent with previous reports (Shibagaki et al., 2002; Yoshimoto et al., 2002).
We determined the root growth capacity of different Arabidopsis lines by comparing the Columbia (Col-0) wild type to 12 Col-0-derived homozygote T-DNA insertion mutant lines, each one bearing a mutation in one of the following sulfate transporter genes: SULTR1;1, SULTR1;3, SULTR2;1, SULTR2;2, SULTR3;1, SULTR3;2, SULTR3;3, SULTR3;4, SULTR3;5, SULTR4;2, SULTR5;1, and SULTR5;2. An additional Col-0 EMS-derived mutant sel1-8 defective in the SULTR1;2 transporter was also studied. This mutant is allelic to the WS-derived T-DNA insertion mutant sel1-11 (Table I). When germinated in vitro on sulfate-free agarose solid growth media containing djenkolate as the sole sulfur source, the roots of all lines grew normally (Fig. 2A ). In the presence of 3 µM of selenate, root growth of the wild-type seedlings stopped following cotyledon emergence as did all the mutants defective in a sulfate transporter, except sel1-8, whose roots continued to grow, reaching approximately 1 cm in length 10 d after germination (Fig. 2A).
SULTR1;2 Mutation Results in an 8-Fold Increase in Selenate Resistance We quantified the selenate resistance of WS and the sel1-11 mutant by establishing the lethal concentration (LC50) curves of root length as a function of selenate concentration in the sulfate-free agarose growth medium. The calculated LC50 value represents the concentration of selenate in the growth medium that causes 50% reduction in root length compared to the control. Seeds were germinated on a solid GM medium containing djenkolate as sole sulfur source and amended with selenate in concentrations ranging from 0 to 50 µM. Seedlings were allowed to grow for 1 week before their root length was measured (Fig. 2B). The calculated LC50 for the wild-type seedlings was 1.5 µM, while that for sel1-11 seedlings was 12.7 µM, which represents an 8-fold increase in selenate resistance of the mutant plants compared to the wild type. Furthermore, the selenate toxicity threshold, which represents the minimal selenate concentration that affects root growth, was less than 0.5 µM for the wild-type seedlings, while sel1-11 seedlings can withstand up to 2 µM of selenate in the external medium without any adverse effect on root growth (Fig. 2B). In the range of higher external concentrations of selenate (20–50 µM), root length of wild-type seedlings was below 10% of that of control plants roots. Although root length of sel1-11 seedlings was markedly reduced, it was never lower than 25% of untreated control plants roots.
Expression of the SULTR1;1 and SULTR1;2 genes was determined by reverse transcription (RT)-PCR in the roots of wild type and sel1-11 mutants in response to sulfate and selenate availability in the culture medium. For this purpose, WS and sel1-11 seedlings were allowed to grow for 8 d on a GM medium containing 1 mM sulfate and then transferred for 2 d to the same medium containing: 1 mM sulfate (+S), no sulfate (–S), or 1 mM sulfate and 0.25 mM selenate (+Se). Plants were then harvested and RT-PCR performed on total RNA extracted from roots (Fig. 3 ).
The SULTR1;2 gene was strongly expressed in both WS and sel1-11 roots independent of sulfate and selenate availability in the medium. The small deletion in the SULTR1;2 gene of sel1-11 did not seem to affect the transcription of the gene or the level of its expression in sel1-11 roots. The SULTR1;1 gene expression in WS roots is repressed when sufficient sulfate is supplied to the plants but increased under sulfate deficiency conditions and by selenate presence in the medium. In sel1-11 roots, SULTR1;1 expression was induced even under sulfate sufficiency conditions, suggesting a functional redundancy in the absence of SULTR1;2 sulfate transport activity.
Because expression of the SULTR1;2 gene was localized primarily in the root epidermis and cortex (Shibagaki et al., 2002
Sulfate and Selenate Accumulation in Roots and Leaves of sel1-11 Are Reduced
Because sulfate uptake by roots of sel1-11 plants was impaired, we investigated sulfate and selenate accumulation in roots and leaves of sel1-11 to determine if they were also reduced. Wild-type and sel1-11 seedlings were allowed to grow for 8 d on a solid GM medium containing 1 mM sulfate as sole sulfur source. Seedlings were then transferred for 2 d to an identical medium containing either 1 mM sulfate (+S), no sulfate (–S), or 1 mM sulfate and 0.25 mM selenate (+Se). Roots and leaves were harvested separately, washed, and their sulfate and selenium contents measured. Selenate could not easily be measured by ionic chromatography due to cosegregation with fumarate, which was also significantly present in leaves. Instead, we routinely measured selenium contents, because we demonstrated, using x-ray absorption spectroscopy (Pickering et al., 2003
Sulfate Uptake Capacity and Selenium Accumulation in Wild-Type and sel1-11 Callus Culture To study the consequences of SULTR1;2 mutation at the cellular level, we used calli produced from WS and sel1-11 seeds by hormone treatment. Selenium accumulation in calli was determined over time after incubation in the presence of 10 µM selenate and djenkolate as sole sulfur source (Fig. 6A ). Selenium accumulation was significantly reduced (75% decrease) in sel1-11 callus compared to wild type after 96 h incubation in presence of selenate.
Sulfate uptake capacity measurements of calli of wild type and sel1-11 lines were performed under the same experimental conditions as for selenium accumulation (Fig. 6B). In the presence of djenkolate as sole sulfur source, sulfate uptake capacity in sel1-11 callus showed a 45% decrease compared to wild type. Addition of 10 µM selenate resulted in a decrease in the sulfate capacity of wild-type calli to reach a level similar of that of sel1-11. These results are consistent with those obtained for whole plants (Figs. 4 and 5).
To test if root growth is a specific target for selenate toxicity, plants were grown normally on a solid GM medium in the absence of selenate for 8 d and then transferred to an identical medium plus or minus 10 µM selenate, in the presence of djenkolate as sole sulfur source. The petri dishes containing the transferred plants were then rotated 180° so the plants were upside down. As shown in Figure 7 , root growth of wild-type seedlings stops after their transfer to a plate containing selenate. Root growth of sel1-11 seedlings continued after transfer to a selenate-containing medium but at a lower rate compared to the nontreated plants (Fig. 7).
Screening an Arabidopsis T-DNA mutant library for selenate resistance enabled us to isolate several resistant mutant lines, which were shown to be alleles of sel1 selenate-resistant mutants deleted in the SULTR1;2 gene (Shibagaki et al., 2002 ). Functional complementation of a yeast mutant strain lacking sulfate transport capacity confirmed that SULTR1;2 encodes a high affinity sulfate transporter (Km approximately 7.4 µM). Furthermore, our results show that the deletion of the SULTR1;2 gene leads to a significant reduction of root sulfate uptake capacity and an impaired response to sulfur deprivation (Fig. 4) and that sulfate accumulation by roots and leaves is markedly reduced in the sel1-11 mutant compared to the wild type (Fig. 5A). This is in agreement with previously obtained results (Shibagaki et al., 2002; Maruyama-Nakashita et al., 2003). Thus, SULTR1;2, which is clearly localized in the outer cell layers of the root as well as in the root tip (Shibagaki et al., 2002; Yoshimoto et al., 2002), appears to play a major role in the acquisition of sulfate by roots of Arabidopsis, and its deletion leads to an inefficient synthesis of downstream sulfur containing amino acids and other metabolites (Maruyama-Nakashita et al., 2003).
In addition to sulfate transport, SULTR1;2 also appears to be involved in the transport of selenate, as suggested by the fact that the mutation of SULTR1;2 in sel1-11 led to a reduction in root and leaf selenium contents (Fig. 5). The role of SULTR1;2 as a major sulfate and selenate transporter is further confirmed by the fact that three different attempts to isolate selenate-resistant mutants through mutant library screening done by our laboratory and two other research teams (Rose, 1997
SULTR1;2 is not the only gene implicated in sulfate acquisition by roots from the soil solution. SULTR1;1 gene, which also encodes a root-expressed high affinity sulfate transporter (Takahashi et al., 2000
SULTR1;1 and SULTR1;2 genes show similar expression patterns under sulfate deficiency (–S) and selenate (+Se) treatment (Fig. 3). Based on similar results obtained in wild-type Arabidopsis plants, it has been suggested that plant selenate treatment might mimic sulfate deficiency (Takahashi et al., 2000
It is noteworthy that no difference is observed in root sulfate uptake capacity of wild-type and mutant plants under selenate treatment. A similar result is obtained in selenate-treated calli (Fig. 6B). This is rather surprising, because the commonly accepted hypothesis attributes the selenate resistance phenotype of the sel1 mutants to a reduced root sulfate uptake capacity compared to wild type, leading to a reduced selenate entry in mutant plants. While a reduction in selenium accumulation is indeed observed in mutant plants compared to the wild type under selenate treatment (Fig. 5B), a comparable root sulfate uptake capacity between both lines is observed under the same conditions. This discrepancy suggests that a more complex determinant confers selenate resistance. The fact that in response to a 48-h selenate treatment, roots of both wild-type and mutant plants showed similar sulfate uptake capacity (Fig. 4) but divergent selenium contents (Fig. 5B) indicated that SULTR1;1 and SULTR1;2 could differ in their affinity for selenate. Because both transporters are active in the roots of wild-type plants and only SULTR1;1 is active in the roots of the mutant sel1-11, we can expect that the affinity of SULTR1;1 for selenate might be lower than that expected for SULTR1;2. If this is the case, then SULTR1;1 activity in sel1-11 roots should be able to maintain a comparable root sulfate uptake capacity to that measured in selenate-treated wild-type plants while transporting less selenate. It has been suggested previously that in wild-type Arabidopsis plants, sulfate transporters might show contrasting selectivities toward sulfate and selenate based on the comparison of sulfate to selenate ratios in the culture medium and in plant tissues (White et al., 2004 The sel1-11 mutant shows enhanced root growth in the presence of toxic external concentrations of selenate (Fig. 2, A and B). The reduced sensitivity of sel1-11 root growth to selenate toxicity is mirrored by an 8-fold increase in LC50 value for selenate and a more than 4-fold increase in the selenate toxicity threshold. Moreover, sel1-11 shows enhanced root growth compared to the wild type even in the presence of relatively high levels of selenate in the medium (up to 50 µM; Fig. 2B). The selenate-resistant phenotype of the sel1-11 mutant could be attributed to a limitation of selenium accumulation in leaves and roots when plants are treated with selenate (Fig. 5B). However, selenate toxicity is generally attributed to the ability of selenate to compete with sulfate for its access to sulfate transporters and to the sulfur metabolic pathway. This would lead to the disruption of sulfur metabolism and to a reduced synthesis of important sulfur-containing compounds, such as Cys, Met, and glutathione, and to the disruption of many cellular processes. Therefore, we propose that selenate toxicity could be better evaluated if sulfate to selenate ratios in tissues are considered instead of absolute selenate contents. Wild-type and sel1-11 selenate-treated seedlings showed a comparable sulfate to selenium ratio in leaves (Fig. 5C). However, sel1-11 roots showed a statistically significant higher sulfate to selenate ratio, suggesting that the selenate resistance mechanism of the sel1-11 mutant might be root specific and consist of the combined effect of a reduced selenate content and a protective role of sulfate against selenate toxicity in roots of the mutant line. The fact that callus cultures showed the same pattern in response to selenate presence in the medium as that of whole plants in terms of selenate accumulation (Fig. 6A) and sulfate uptake capacity (Fig. 6B) suggested that the selenate resistance mechanism in sel1-11 takes place at the cellular level rather than the tissue or organ level.
The activity of the rapidly dividing cells in the root meristem is vital for root growth, and we propose that the root apical region might be the target of selenate toxicity in roots. This view is supported by the fact that SULTR1;2 gene promoter fused to The isolation and characterization of a selenate-resistant mutant, sel1-11, enabled the identification of the SULTR1;2 gene as a major contributor in the acquisition of not only sulfate but also of selenate by Arabidopsis roots. We revealed the peculiarity of SULTR1;2 in Arabidopsis, demonstrating that it is, among 13 mutated sulfate transporters, the only one whose mutation conferred resistance to selenate. We therefore highlighted that SULTR1;2 function is not fully redundant, neither with its root-localized structural homolog SULTR1;1 nor with 12 other putative Arabidopsis sulfate transporters. The analysis of the selenate-resistant phenotype of the sel1-11 mutant suggests that root growth, particularly activity at the root tip, might be a major target of selenate toxicity in Arabidopsis.
Plant Material Arabidopsis (Arabidopsis thaliana) L. Heynh ecotype WS was used in this study, and the population of T-DNA mutants (around 30,000 lines) were provided by the Station de Génétique et d'Amélioration des Plantes of the Institut National de la Recherche Agronomique, Versailles, France. Seeds of T-DNA insertion Col-0 mutant lines defective in sulfate transporters were provided by Max Planck Institut sultrl1-3 line (121G06) and by the Arabidopsis Biological Resource Center (Ohio State University) sultr1-1 (SALK-093256); sultr2-1 (SALK-109907); sultr2-2 (SALK-054730); sultr3-1 (SALK-023190); sultr3-2 (SALK-023980); sultr3-3 (SALK-031340); sultr3-4 (SALK-100362); sultr3-5 (NASC-N112372); sultr4-2 (SALK-103827); sultr5-1 (SALK-015044); and sultr5-2 (SALK-118311). EMS-mutagenized seeds of sel1-8 (Col) line were kindly provided by Dr. N. Shibagaki (Carnegie Institution Washington, Palo Alto). The mutant sultr4-1 was not found in seed stock center during the time span of this work.
Hydroponic Cultures
In Vitro Cultures
Callus Culture
Seeds of 30,000 T-DNA insertion mutant lines were surface-sterilized, sown, and grown in vitro for 2 weeks on an agarose GM containing 10 µM selenate and 100 µM djenkolate as the sole sulfur source. Seedlings, whose roots were at least 5 mm long, were selected and transferred for recovery to a nonselective Murashige and Skoog medium lacking selenate for 1 week; they were then transferred to peat and allowed to self fertilize. Seeds from the candidate lines were grown in vitro on agarose for an additional identical screening in the presence of 10 µM selenate.
Soluble anions were extracted by incubating 50 mg of fresh weight plant material in 2 mL of deionized water and heated at 70°C for 30 min. Sulfate, as well as major inorganic and organic anions, was analyzed by high performance ionic chromatography (LC20, Dionex) using an IonPaq AS11 column and a sodium hydroxide (1–22 mM) linear gradient. Plant samples for selenium speciation were immediately kept on dry ice after harvest, then ground in liquid nitrogen, and prepared as previously described for Se K-edge x-ray absorption spectroscopy (Pilon-Smits et al., 1999
Plants were grown vertically in petri dishes on appropriate selective media. Roots were regularly scanned using an Epson Perfection 1240U scanner and their length measurements carried out using the OPTIMAS software (OPTIMAS Image Analysis system 6.1, Media Cybernetic). Data were analyzed using the SigmaPlot 7 analysis software (SPSS).
Plants were harvested, rapidly weighed, then flash frozen in liquid nitrogen and stored at –80°C until extraction. RNA and DNA were extracted using the RNeasy Plant Mini kit (Qiagen) and the GeneElute Plant Genomic DNA kit (Sigma), respectively, following the manufacturer's instructions.
The cDNA first-strand synthesis was done using the Omniscript reverse transcriptase (Qiagen). DNA amplifications by PCR were done using Taq DNA Polymerase (Invitrogen). Thirty cycles were generally used during PCR unless otherwise indicated. Actin was used to normalize cDNA loading. The following primers were used to amplify the corresponding genes: SULTR1;1F (5'-CTACAGTATCCGGACGCTGCCCAAA-3'); SULTR1;1R (5'-CGATATTAAGTTTGTTGCTCAGCCACTTCC-3'); SULTR1;2F (5'-AGCAGCAAGCCTACCTAGGATTCA-3'); SULTR1;2R (5'-CTTGACCCCTTGGTGTGATAGAAGAATC-3'); ActinF (5'-GGTAACATTGTGCTCAGTGGTGG-3'); and ActinR (5'-AACGACCTTAATCTTCATGCTGC-3').
The genomic sequence of the SULTR1;2 gene was amplified by PCR using a Pfu high fidelity DNA polymerase (Promega) and the following primers: forward primer SULTR1;2F2 (5'-CCTGGTTCGATACCATTACTCCATCCAC-3'); and reverse primer SULTR1;2R2 (5'-CTTGACCCCTTGGTGTGATAGAAGAATC-3'). This fragment was cloned in the pCR-Script Amp SK(+) plasmid (Stratagene) at the Srf1 restriction site. The sequencing was done by Genome Express.
The yeast (Saccharomyces cerevisiae) strain used is YSD1 (Smith et al., 1995
Sulfate influx measurements were performed using whole plants grown hydroponically for 4 weeks. Roots of whole plants were placed in a 100-µM K2SO4 solution at pH 5.0 (adjusted with MES buffer), aerated, and thermoregulated at 25°C for 10 min and then transferred in an identical solution and pulsed for an additional 10 min in the presence of 0.03 µCi/mL (1.12 kBq/mL) of the radiotracer 35S-SO4 (Amersham Biosciences). The roots were then washed for 1 min in an ice-cold K2SO4 5 mM solution, harvested, blotted with paper towel, and their soluble contents extracted in a 100-mM HCl solution heated for 30 min at 70°C. The radioactivity of an aliquot of the acid extract was determined after addition of a scintillation liquid (Ultima gold, Packard), using a Sulfate influx measurements were performed using 4-week-old calli using similar conditions as described for root measurements, except that the calli were handled using a vacuum filtration device.
ANOVA was used for statistical analysis of the data. Mean separation procedures were carried out using the multiple range tests with Fisher's LSD procedure (P < 0.05).
We thank Dr. Franck W. Smith (Commonwealth Scientific and Industrial Research Organization, St. Lucia, Australia) for the kind gift of the yeast mutant YSD1. Technical expertise of Cécile Lambert (Biochimie et Physiologie Moléculaire des Plantes, Montpellier, France) in gene mapping has been very much appreciated. Received October 18, 2006; accepted December 26, 2006; published January 5, 2007.
1 This work was supported by the French research organizations (Institut National de la Recherche Agronomique and Centre National de la Recherche Scientifique), by Génoplante (grant no. AF 1999–061 to J.-C.D.), by the France-Berkeley Foundation (grants to J.-C.D. and N.T.), by the French Ministère de l'Enseignement Supérieur et de la Recherche (fellowship to E.E.K.), and by the Tunisian government (fellowship to H.R.). 
2 Present address: Center for Rhizosphere Biology, Department of Horticulture, Colorado State University, Fort Collins, CO 80523–1173.
3 Present address: Laboratoire de Biotechnologie des Plantes, Université de Lausanne, CH–1015 Lausanne, Switzerland. The author responsible for distribution of materials integral to the findings presented in this article in accord with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jean-Claude Davidian (davidian{at}supagro.inra.fr).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.091462 * Corresponding author; e-mail davidian{at}supagro.inra.fr; fax 33–0467525737.
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TOP
ABSTRACT
RESULTS
5 mm were selected for further analysis; under these conditions, root lengths of wild-type plants were no longer than 2 mm. From 30,000 T-DNA mutagenized T2 lines, 132 putative selenate-resistant mutants were isolated. After rescreening the progeny of the putative mutants, the selenate-resistant phenotype was confirmed for only four mutant lines: A25, B78, B181, and A251. 
-glucuronidase reporter gene was clearly shown to be localized in the root tip and the root cap (Shibagaki et al., 2002