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First published online September 17, 2004; 10.1104/pp.104.046441 Plant Physiology 136:3396-3408 (2004) © 2004 American Society of Plant Biologists Regulation of Sulfate Uptake and Expression of Sulfate Transporter Genes in Brassica oleracea as Affected by Atmospheric H2S and Pedospheric Sulfate Nutrition1Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom (P.B., M.J.H.); Laboratory of Plant Physiology, University of Groningen, 9750 AA Haren, The Netherlands (C.E.E.S., S.W., L.J.D.K.); and Institute of Plant Science, University of Heidelberg, 69120 Heidelberg, Germany (M.W., R.H.)
Demand-driven signaling will contribute to regulation of sulfur acquisition and distribution within the plant. To investigate the regulatory mechanisms pedospheric sulfate and atmospheric H2S supply were manipulated in Brassica oleracea. Sulfate deprivation of B. oleracea seedlings induced a rapid increase of the sulfate uptake capacity by the roots, accompanied by an increased expression of genes encoding specific sulfate transporters in roots and other plant parts. More prolonged sulfate deprivation resulted in an altered shoot-root partitioning of biomass in favor of the root. B. oleracea was able to utilize atmospheric H2S as S-source; however, root proliferation and increased sulfate transporter expression occurred as in S-deficient plants. It was evident that in B. oleracea there was a poor shoot to root signaling for the regulation of sulfate uptake and expression of the sulfate transporters. cDNAs corresponding to 12 different sulfate transporter genes representing the complete gene family were isolated from Brassica napus and B. oleracea species. The sequence analysis classified the Brassica sulfate transporter genes into four different groups. The expression of the different sulfate transporters showed a complex pattern of tissue specificity and regulation by sulfur nutritional status. The sulfate transporter genes of Groups 1, 2, and 4 were induced or up-regulated under sulfate deprivation, although the expression of Group 3 sulfate transporters was not affected by the sulfate status. The significance of sulfate, thiols, and O-acetylserine as possible signal compounds in the regulation of the sulfate uptake and expression of the transporter genes is evaluated.
In general plants utilize sulfate taken up by the roots as the main S source for growth (Cram, 1990  ; Clarkson et al., 1993) and sulfate is reduced to sulfide before it is assimilated into organic S compounds. Under normal conditions the rate of uptake and assimilation of S will depend on the requirement for growth, which can be defined as the rate of S uptake and assimilation required per gram plant biomass produced with time (De Kok et al., 2002). The S requirement will fluctuate during plant development and may vary between species differing in S need for growth and the potential sink capacity of secondary S compounds.
The uptake of sulfate by the roots and its transport to the shoot appear to be major sites of regulation of S assimilation (Hawkesford and Wray, 2000
Sulfate transporter genes have been identified from Arabidopsis and other plant species (Smith et al., 1995
There is little information on shoot-root coordination of the uptake of sulfate versus its assimilation and the signal transduction pathways involved. There may be fast responses to environmental change, for instance S supply, via metabolite activation and deactivation of enzymes involved in S assimilation (Hell et al., 2002
Plants are able to utilize forms of S other than sulfate taken up from the pedosphere such as foliarly absorbed atmospheric H2S, which is directly metabolized into Cys and subsequently into other organic S compounds, including glutathione and proteins (De Kok, 1990 In this paper the modulation of S nutrition of B. oleracea was achieved by changing the levels of atmospheric H2S and pedospheric sulfate in order to probe the regulation of sulfate uptake and transport as indicated by the expression of the respective genes in relation to the S status of the plant. The expression patterns of the complete Brassica sulfate transporter gene family are presented for roots, stem, and leaves. In addition, analysis of sulfate, thiols, total S, and O-acetylserine (OAS) was examined to determine the relationship of these metabolite pools to the expression of the sulfate transporter gene family in uptake and movement of sulfate within the plant.
Impact of Sulfate Deprivation and H2S Exposure on Growth, Sulfur, and Nitrogen Metabolites and Sulfate Uptake Capacity
H2S is a potentially phytotoxic gas; however, B. oleracea is not very susceptible to H2S and its growth is only significantly reduced at atmospheric levels higher than 400 nL L–1 (De Kok et al., 1997
As has been observed previously, the transfer of B. oleracea seedlings to sulfate-deprived conditions resulted in a rapid development of S deficiency symptoms from approximately 6 d onwards (Table I; De Kok et al., 1997
Upon sulfate deprivation, the appearance of S deficiency symptoms was preceded by a rapid decrease in the sulfate content in all plant parts, which was already significant after 1 d (Fig. 1). However, after 6 d, when plants started to show visible symptoms of sulfate deficiency, all plant parts still contained detectable amounts of sulfate (Fig. 1). In sulfate-deprived, H2S-exposed plants, the sulfate contents in leaves, stems and roots were as low as in sulfate-deprived nonexposed plants.
Sulfate deprivation resulted in a decrease in the water-soluble nonprotein thiol content, however to a lesser extent than for sulfate (Fig. 1). H2S exposure generally results in an increased size of the water-soluble nonprotein thiol pool (predominantly glutathione) in the shoot, since the absorbed H2S is metabolized into Cys with high affinity and subsequently into glutathione and other S metabolites (De Kok et al., 1997 , 2000, 2002). Indeed, in sulfate-sufficient, H2S-exposed plants the thiol content in leaves was enhanced up to 1.4-fold after 1 d of exposure and it was hardly affected during further exposure. The thiol contents of the roots and stem plus petioles of sulfate-sufficient plants were not affected by H2S exposure (Fig. 1). However, in sulfate-deprived, H2S-exposed plants the total water-soluble nonprotein thiol content in leaves was increased 1.5-fold after 1 d of H2S exposure and further increased with time reaching a level similar to that in leaves of sulfate-sufficient, H2S-exposed plants (Fig. 1). From day 1 to 3, the thiol content in the stem and roots of sulfate-deprived, H2S-exposed plants decreased by nearly the same extent as for sulfate-deprived plants. However, from day 6 onwards the thiol content of both, stem and roots of sulfate-deprived, H2S-exposed plants remained unaltered (Fig. 1). Ten days of sulfate deprivation resulted in a 4- and 8-fold decrease in total S content of roots and shoot, respectively; it did not affect the total N content, which resulted in a strongly increased N:S ratio (Table III). It was striking that the total S content was hardly affected in sulfate-deprived, H2S-exposed plants as compared to that of sulfate-deprived nonexposed plants (Table III). The total N content was not affected by H2S exposure and as a consequence the total N/S ratio remained extremely high as compared to that of sulfate-sufficient plants (Table III).
Brassica originates from saline and S enriched environments and is considered to have a high S requirement for growth (Westerman et al., 2001a ). However, in Brassica seedlings a large proportion of total S (up to 90%) may be present as sulfate, presumably located in the vacuole and here the concept of "the S need for structural growth" may have to be redefined (Castro et al., 2003). From the present data it is evident that sulfate-deprived, H2S-exposed plants were able to grow nearly optimally and quite efficiently at a minimum S level (Table III).
The depletion of S-containing amino acids and a subsequently restricted protein synthesis is likely the primary cause of the disturbance of physiological functioning of the plant and the appearance of S deficiency symptoms. The development of S deficiency symptoms is generally accompanied by accumulation of nitrate, free amino acids (especially in the shoots), and an increase in dry matter content due to an accumulation of nonstructural carbohydrates such as starch (De Kok et al., 1997
Despite the fact that sulfate deprivation did not affect total N content (Table III), it had a decisive impact on the content of several N metabolites. For instance, the content of OAS, which is the direct carbon/N precursor for Cys synthesis, is assumed to have significance in the regulation of sulfate uptake and reduction (Smith et al., 1997
Similarly to previous observations, sulfate deprivation resulted in a rapid increase in the sulfate uptake capacity (Fig. 3; Clarkson et al., 1993 ; Hawkesford and Smith, 1997). The sulfate uptake capacity increased 1.4-fold after 1 d of sulfate deprivation and it slightly further increased with time upon deprivation up to 1.6-fold after 10 d. The nitrate uptake capacity, however, was decreased from 6 d onwards down to 0.5-fold (to 0.23 mmol g–1 root fresh weight d–1) after 10 d of sulfate deprivation (data not shown). Even when H2S was used as the S source and growth of sulfate-deprived plants was (except root growth) quite similar to that of sulfate-sufficient plants, the enhanced sulfate uptake capacity upon sulfate deprivation was not affected by H2S exposure (Fig. 3). The sulfate uptake capacity of sulfate sufficient plants was hardly affected by H2S exposure.
Isolation and Identification of the Brassica Sulfate Transporter Family
Plants contain a variety of sulfate transporters with specific functions for the uptake of sulfate by the roots, transport to the shoot, and subcellular distribution (Hawkesford and Smith, 1997 The use of degenerate primers based on the high identity sequence regions of known plant sulfate transporter groups (Table IV) as well as Brassica genomic sequence information enabled the isolation by PCR and the identification of 11 sulfate transporter cDNAs from B. napus and B. oleracea. Sequence comparison and phylogenetic analysis of the coding regions of the isolated Brassica sulfate transporter mRNAs to the known sulfate transporter family from Arabidopsis indicated that three transporters, BSultr1;1, 1;2, 1;3, belong to Group 1, one, BSultr 2;1, belongs to Group 2, five, BSultr 3;1, 3;2, 3;3, 3;4 and 3;5 belong to group 3 and two, BSultr4;1 and 4;2, belong to the Group 4 sulfate transporter family (Fig. 4). The sequence similarity of the coding mRNA region of the Brassica sulfate transporters to the homologous Arabidopsis genes was in the range of 84% to 89%.
There is a close phylogenetic relationship between Arabidopsis and Brassica species, which was evident from the high sequence identities between the Brassica and the homologous Arabidopsis sulfate transporter mRNAs including the subdivision into 4 groups (Fig. 4). To date proton-coupled sulfate transport activity has been demonstrated only for Group 1 and 2 transporters; however, because of the high homology between the members of the family, it is reasonable to expect that most are involved in sulfate transport (Hawkesford, 2000 ). The close phylogenetic relationship between Arabidopsis and Brassica species, and the high sequence similarities of the sulfate transporters suggest a similar if not identical function of the Brassica sulfate transporter homologs.
The expression pattern in B. oleracea roots indicated that there was no need for expression of all sulfate transporters under sufficient sulfate supply. As in Arabidopsis, the abundant expression of BSultr1;2 (Fig. 5) suggested that the 1;2 transporter is mainly responsible for the initial uptake of sulfate (Takahashi et al., 2000
To our knowledge, this is the first study reporting Group 3 sulfate transporter expression in roots. The highest transcript abundances were seen for BSultr3;2, 3;3, and 3;5 (Fig. 5). In Arabidopsis transcripts of Sultr3;1, 3;2, and 3;3 were detected in leaves but not in roots (Takahashi et al., 2000 ). Whether the Brassica Group 3 sulfate transporters fulfill a novel function is speculative without any cellular localization data of the different members of Group 3. There was also a high expression of the Group 4 Sultr4;1 in B. oleracea roots. The expression of a sulfate efflux transporter in the tonoplast, as attributed to this group, would limit accumulation of sulfate in the vacuole. Although the nature of the vacuole influx transporter is not known yet, the expression of a vacuole efflux transporter in roots would favor transport of sulfate in the direction of vascular long distance transport rather than vacuole accumulation for storage. The stem is the major aerial support system in most plants and provides a pathway for transport between the shoot and the root. The exclusive Sultr2;1 expression in the stem contrasted with the expression of Sultr2;2 in the root (Fig. 6). The role of a vascular sulfate transporter in the stem under sufficient S supply would be to support sulfate distribution from the vascular tissue to the surrounding parenchyma tissue for growth and maintenance compared to the role in the root which would be to take up sulfate for vascular transport. The weak expression of the Group 4 transporter in the stems (Fig. 6) might suggest a similar role as for BSultr4;1 in roots. There was no change in the sulfate content in stems visible under sufficient sulfate indicating reduced or no vacuolar sulfate accumulation. Compared to the roots a change of expression pattern was also found for the Group 3 transporters. In stems, BSultr3;1, 3;3, and 3;4 were the prominent Group 3 transporter transcripts (Fig. 6), which might indicate tissue or organ specific function of the different Group 3 transporters.
Leaves are the major site of sulfate assimilation. If sufficient sulfate was supplied to B. oleracea, sulfate accumulation was found in leaves, which apparently was not needed for assimilation and growth. Apart from the expression of the Group 2 Sultr 2;1, there was only clear expression of the Group 3 transporter, Sultr3;3, and a weak expression of Sultr3;1 in the leaves (Fig. 7). This result would indicate the need for high expression of only three transporters for plasma membrane proton/sulfate cotransport in the entire leaf: BSultr2;1 for the vascular cellular uptake of sulfate delivered via xylem in vascular tissues and BSultr3;3 and 3;1 for cellular uptake of sulfate into nonvascular parenchyma cells. These results differed from the expression pattern found in Arabidopsis leaves, in which expression of both Group 2 transporters as well as of Sultr4,1 and the Group 3 transporters Sultr3;1, 3;2, and 3;3 were detectable by reverse transcription (RT)-PCR (Takahashi et al., 2000 ).
Expression of Sulfate Transporters upon Sulfate Deprivation and H2S Exposure The transfer of the plants to sulfate-deprived conditions resulted in a drastic change of the expression pattern of the Group 1, 2, and 4 sulfate transporters in the roots (Fig. 5). After 1 d of sulfate deprivation an up-regulation and induction, respectively, of expression was found for Group 1 BSultr1;1, 1;2, and both Group 4 transporters. An induction of expression was also noticeable for BSultr1;3 but with a lower intensity than for BSultr1;1 and 1;2 (Fig. 5). The increase of the Group 2 transporter mRNA abundance occurred only at day 3 of deprivation, with a highest peak of transcript abundance for all Group 1, 2, and 4 transporters detected at day 6 of sulfate deprivation. In parallel to the reduction of uptake capacity with further sulfate depletion, all transcript abundances were diminished by day 10 (Fig. 5). H2S exposure did not alter the sulfate deprivation induced coordinated expression of the Group 1, 2, and 4 transporters in roots. A slightly reduced transcript signal was seen for BSultr1;1 and 4;2 under H2S treatment, which did not result in a reduction of the sulfate uptake capacity (Figs. 3 and 5). In contrast, the expression of the Group 3 sulfate transporters in the roots was not influenced by the S status of the plants, either by H2S exposure or by sulfate deprivation (Fig. 5). A differential expression pattern of the sulfate transporter genes was also detectable in stem tissues (Fig. 6). The Group 1 sulfate transporters were not expressed in the stem except after 10 d of sulfate deprivation, when there was a small induction of BSultr1;1 transporter expression. In contrast to the expression in the roots, the Group 2 BSultr2;1 transporter expression was up-regulated; however, no induction of BSultr2;2 expression occurred in stems of plants by sulfate deprivation (Fig. 6). Sulfate deprivation resulted in an increase of BSultr4;1 and BSultr4;2 transcript with the duration of deprivation (Fig. 6). H2S exposure repressed BSultr1;1 expression, but otherwise had no effect on BSultr4;1 and 4;2 expression (Fig. 6). In leaves, from day 1 of sulfate deprivation onwards, an induction of both Group 4 transporters occurred and transcript abundance increased with the duration of the sulfate deprivation. In addition, after 6 d of sulfate deprivation, expression of BSultr1;1 and BSultr1;2 was induced (Fig. 7). H2S exposure reduced the expression pattern of the Group 1 and 4 transporters in sulfate-deprived leaves (Fig. 7). Both in roots and stems, the transcript abundances of the leaf expressed transporters BSultr3;1 and BSultr3;3 were not altered by changing the S status (Fig. 7).
The expression analysis of all of the Brassica sulfate transporters indicated that the sulfate transport system can be split into nutritionally regulated and nonregulated parts. The regulated system is represented by the Group 1, 2, and 4 transporters and the nonregulated system by the Group 3 transporters.
When the sulfate supply was removed, the reduction of the root sulfate concentration was concomitant with an increase of the total root sulfate uptake capacity (Figs. 1 and 3). The increased transporter activity in plasma membrane vesicles isolated from sulfate-deprived roots (Hawkesford et al., 1993 Although large increases of both Group 1 sulfate transporter transcripts were observed, the total root sulfate uptake capacity was only approximately doubled during sulfate deprivation. This might be an indication of further posttranscriptional regulation of Group 1 transporters. Although the signal intensities differed drastically, all genes were coordinately controlled. After 1 d of sulfate deprivation, derepression occurred with increasing transcript abundance to day 6, followed by a coordinated reduction of transcript at day 10. This coordinated reduction of expression may be a consequence of alteration of the root structure indicated by the reduced shoot to root ratio in S deprived H2S nonfumigated as well as fumigated plants. Based on the putative function of the Group 1, 2, and 4 transporters, this derepression under S stress will maximize uptake, vacuole efflux, and vascular transport of sulfate to the growing shoot. Additionally the induction and/or up-regulation of Sultr1;3 and the Group 2 transporters may fulfill a dual function for translocation of sulfate from the phloem to the root derived from upper source organs by remobilization. Group 3 sulfate transporter expression was not regulated by the S nutritional status of the plant. As there was no expression of Group 3 transporters found in Arabidopsis roots, an involvement of the Group 3 transporters in the initial uptake of sulfate is unlikely. The contrasting expression pattern in B. oleracea roots compared to Arabidopsis suggests a need for further analysis of the Group 3 sulfate transporters to elucidate the importance of this nonregulated system in sulfate transport.
In the upper parts of the plant, the nutritional regulated sulfate transport system followed a dual pattern: an early and a late response on sulfate deficiency. One of the early responses in stems and leaves is the up-regulation in stems, and induction in leaves, of both Group 4 transporters. Increased expression of Group 4 sulfate transporters would favor efflux of sulfate from the vacuoles to maintain the supply of sulfate for assimilation and remobilization of sulfate from leaves for transport to other organs. Up-regulated expression of Sultr2;1 would strengthen the cellular uptake of xylem derived sulfate in the vascular tissue. The export of sulfate by source leaves, especially under sulfate stress, would require changes in the expression of transporters involved in vascular transport especially for phloem loading and/or retransportation into xylem. Interestingly, none of the regulated Arabidopsis genes expressed in the vascular tissue were influenced by sulfate deprivation in the B. oleracea leaves. In Arabidopsis Sultr2;1 expression was down-regulated, but in B. oleracea no change of expression of Sultr2;1 was visible. The expression of AraSultr2;2 and 1;3 was up-regulated in Arabidopsis leaves under sulfate deficiency (Takahashi et al., 2000
In oilseed rape an export of sulfate from older leaves was found after 6 d of sulfate deprivation (Blake-Kalff et al., 1998 The expression of Group 3 transporters in the stem and leaves suggested a need for the constitutive, nonregulated sulfate transport system. Differences in the expression pattern of the individual Group 3 transporters suggest specific functions in the respective tissues.
Sulfate uptake and transport is dependent on the S-nutritional status of the plant. In addition activities of enzymes involved in sulfate reduction and assimilation are regulated (Leustek and Saito, 1999
The up-regulation of the transporters is much reduced in response to S deficiency when N is limiting (Clarkson et al., 1989
From the present data it is obvious that the shoot to root signaling of the regulation of the sulfate uptake capacity in B. oleracea roots is unlikely to be mediated by the size of the thiol pool (Westerman et al., 2000a
This study confirmed that sulfate deprivation of B. oleracea seedlings induced multiple responses, which may facilitate increased sulfate uptake efficiency. Initially, there was a rapid induction of the sulfate uptake capacity by the roots, accompanied by an expression of genes encoding sulfate transporters in the roots and in the rest of the plant. Second, upon more prolonged sulfate deprivation there was a change in shoot and root biomass partitioning in favor of root production. Indirect evidence suggested that sulfate itself has the potential to be involved in sensing or as a regulatory signal in the roots, while the role of OAS and reduced thiols such as glutathione remains unclear. However, it is evident that in B. oleracea there was a poor shoot to root signaling in the regulation of sulfate uptake and expression of the sulfate transporters.
Plant Material Brassica oleracea L. cv Arsis (Royal Sluis, Enkhuizen, The Netherlands) was germinated in vermiculite in a climate-controlled room. Day and night temperatures were 21°C and 17°C, respectively, relative humidity was 60% to 70%, and the photoperiod was 14 h at a photon fluence rate of 250 to 300 µmol m–2 s–1 (within the 400–700 nm range).
Ten-day-old seedlings were transferred to a 25% Hoagland nutrient solution (pH 6) at 0.5 mmol L–1 For analysis of the expression of sulfate transporters, plant material was frozen in liquid N2 and stored at –80°C until further use.
Plants were exposed in 150-L cylindrical double-walled stainless steel exposure cabinets (0.6 m diameter) with a polycarbonate top. Day and night temperatures were 20°C and 16°C (±1°C), respectively, relative humidity was 40% to 50%, and the photoperiod was 14 h at a photon fluence rate of 320 to 350 µmol m–2 s–1 at plant height. The air exchange in the cabinets was 40 L min–1 and the air inside was stirred continuously by two ventilators (air exchange capacity of 59 m3 h–1 each) placed on the bottom. Pressurized H2S diluted with N2 (1 mL L–1) was injected into the incoming air stream and adjusted to the desired concentration by AMS electronic mass flow controllers (Bilthoven, The Netherlands). The H2S concentration in the cabinets was controlled by an SO2 analyzer (model 9850) equipped with an H2S converter (model 8770, Monitor Labs, Measurement Controls, Englewood, CO).
Analysis of total S content was performed according to a modification of the method of Jones (1995)
The total N content was determined with a modified Kjeldahl method. Plant tissue was dried at 100°C for 24 h, pulverized by a Retsch Mixer-Mill (type MM2; Haan, Germany), and 50 mg was weighed on a small ash free filter. To the filter 0.33 g Na2S2O3.5H2O and a small spoon of catalyst (K2SO4:CuSO4:Na2SeO3, 15:5:0.085 [w/w/w]) were added; the filter was folded and transferred into a destruction tube. To each tube 2 mL of destruction solution (33.3 g Na salicylate in 1 L H2SO4 [95%–97%]) was added and incubated overnight. The tubes were placed in a Kjeldatherm-Digestion unit (type KB 40 S, Gerhardt, Bonn) coupled with a Variostat temperature-time-programmer (Gerhardt) and the temperature was very slowly increased up to 250°C, then small funnels were placed on top, and then the temperature was further increased to 360°C. At this temperature the samples were boiled for 3 h until the solution was clear and subsequently cooled down overnight. The tubes were heated up to 100°C, the nondestructed particles were washed down from the wall, the temperature was raised to 360°C again, and the samples were boiled for a short time. After cooling down, the samples were quantitatively transferred to 50-mL flasks with demineralized water and made up to 50 mL. NH4+ was determined colorimetrically at 410 nm after reaction with Nessler's reagent.
For measurement of the
Plant tissue was homogenized in 80 mmol L–1 sulfosalicylic acid, 1 mmol L–1 EDTA, and 0.15% (w/v) ascorbic acid with an Ultra Turrax (Polytron pt 3000, Kinematica AG, Littau, Switzerland) at 0°C (1 g fresh weight in 10 mL). Oxygen was removed from the solution by N2 bubbling. The homogenate was filtered through one layer of Miracloth (Calbiochem Corporation, LaJolla, CA) and the filtrate was centrifuged at 30 x 103 g for 15 min (0°C). Total water-soluble nonprotein thiol content was determined colorimetrically at 412 nm after reaction with 5,5'-dithiobis[2-nitrobenzoic acid] according to De Kok et al. (1988)
Plant tissue was freeze-dried in a Heto LyoLaB 3000 freeze dryer (Heto-Holten A/S, Allerød, Denmark) and 100 mg tissue material was ground in liquid N2 to a fine powder and extracted with 1 mL 0.1 M HCl for 15 min at 4°C while shaking. Homogenates were centrifuged twice at 15 400g, 4°C for 5 min. The resulting supernatants were used for further analysis. Determination of OAS was based upon derivatization with the fluorescence dye AccQ-Tag (Waters, Milford, MA). An appropriate volume of supernatant (5–20 µL) was derivatized according to the manufacturer (Manual WAT052874TP). The resulting OAS derivative was separated from other amino acid derivatives by reverse phase HPLC as described in Wirtz et al., 2004
For measurement of the
Total RNA from roots, stems, and leaves was isolated by a method based on Verwoerd et al. (1989)
Partial cDNAs corresponding to the 11 sulfate transporter genes were isolated by RT-PCR from RNA from Brassica napus and B. oleracea. Degenerate primers were designed based on highly homologous regions identified in sequence alignments of published sulfate transporter genes of the different sulfate transporter subfamilies 1 to 4 (Table IV). For the Group 1 BSultr 1;3, Group 3 BSultr 3;2, 3;3, 3;4, 3;5, and the Group 4 BSultr 4;2 sulfate transporter partial gene sequence information was obtained via BLAST search using Arabidopsis sulfate transporter sequence information (data not shown) on The Brassica oleracea Genome Database at the Institute for Genomic Research in Rockville, Maryland. Oligonucleotide primers were generated (Table IV) to isolate the corresponding cDNAs via 3'-RACE approach. First strand cDNA synthesis was performed according the Invitrogene Superscript II Reverse Transcriptase manual with 1-µg aliquots of total root and leaf RNA and antisense primer. Following PCR was performed according the Promega Taq-DNA polymerase standard protocol for 50 µL reaction by using a 1-µL aliquot of the each first strand cDNA solutions and specific sense/antisense primer combinations. 5'- and 3'-region of the sulfate transporter transcripts were isolated by 5'- and 3'-RACE approach according the Invitrogene 5'-RACE and 3'-RACE kit manual and sequence specific oligonucleotide primers based on the sequence results from the partial cDNA fragment isolation (data not shown). Finally the full-length cDNA containing the full coding region were generated via RT-PCR and sequence specific primer obtained from the 5'- and 3'-RACE fragments (data not shown) using proofreading Pfu-DNA polymerase in the PCR reaction. All PCR fragments derived from RT-PCR or RACE were verified by electrophoresis, gel-eluted according the Qiagen QIAquick gel extraction kit manual, ligated into the Promega pGEM-Teasy vector system, and sequenced in both directions.
DNA sequences were determined by PCR based the dideoxy method on an Applied Biosystems 377 DNA sequencer or a Perkin Elmer ABI Prism 310 genetic analyzer. ClustalX V.1.81 (Thompson et al., 1997
Northern hybridization of the described Brassica sulfate transporter transcripts was performed according Church and Gilbert (1984) Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AJ416460, AJ311388, AJ633707, AJ633705, AJ581745; AJ601439, AJ704373, AJ704374, AJ633706, AJ416461, AJ555124. Received May 14, 2004; returned for revision June 9, 2004; accepted June 14, 2004.
1 This work was supported by the Biotechnology and Biological Science Research Council (BBSRC), UK.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.046441. * Corresponding author; e-mail peter.buchner{at}bbsrc.ac.uk; fax 44–1582–763010.
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ABSTRACT
RESULTS AND DISCUSSION
(+S) and grown for 7 d. Subsequently, plants were transferred to stainless steel containers containing a 25% Hoagland solution at 0 mmol L–1 
At the beginning of the uptake measurements and after 24 h, the nutrient solution was adjusted to the original volume by weighing. Aliquots were taken from the nutrient solution and the 
