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First published online August 24, 2007; 10.1104/pp.107.105742 Plant Physiology 145:378-388 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Posttranscriptional Regulation of High-Affinity Sulfate Transporters in Arabidopsis by Sulfur Nutrition1,[OA]RIKEN Plant Science Center, Tsurumi-ku, Yokohama 230–0045, Japan (N.Y., E.I., A.W.-T., K.S., H.T.); and Graduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263–8522, Japan (K.S.)
High-affinity sulfate transporters SULTR1;1 and SULTR1;2 are expressed at epidermis and cortex of Arabidopsis (Arabidopsis thaliana) roots during sulfur limitation. Here, we report that SULTR1;1 and SULTR1;2 are two essential components of the sulfate uptake system in roots and are regulated at posttranscriptional levels together with the previously reported transcriptional control. Double knockout of SULTR1;1 and SULTR1;2 by T-DNA insertion gene disruption resulted in complete lack of sulfate uptake capacity and severely affected plant growth under low-sulfur conditions. Expression of epitope-tagged proteins SULTR1;1mycHis and SULTR1;2mycHis, under the control of the cauliflower mosaic virus 35S promoter, rescued the uptake of sulfate and the growth of the sultr1;1 sultr1;2 double knockout mutant. The recovery of the double knockout phenotypes was attributable to the posttranscriptional accumulation of sulfate transporter proteins that derive from the epitope-tagged transgenic constructs. Both SULTR1;1mycHis and SUTLR1;2mycHis mRNAs were predominantly found in roots and slightly induced by long-term sulfur limitation. SULTR1;1mycHis and SULTR1;2mycHis proteins were found exclusively in roots, and significantly accumulated by sulfur limitation, correlating with the induction of sulfate uptake activities. In the time course of short-term sulfate starvation treatment, SULTR1;1mycHis and SULTR1;2mycHis proteins were significantly accumulated during the 8- to 72-h period, causing substantial induction of sulfate uptake activities, while their corresponding mRNAs were expressed constantly around the initial levels, except for the transient induction in the first 2 h. This study suggested the importance of root-specific and sulfur deficiency-inducible accumulation of SULTR1;1 and SULTR1;2 sulfate transporter proteins for the acquisition of sulfate from low-sulfur environment.
Sulfur is a macronutrient required for plant growth. Plants usually assimilate the oxidized form of sulfur, sulfate anion, to generate essential sulfur-containing amino acids (Leustek et al., 2000  ; Saito, 2004). The plasma membrane-bound sulfate transporters mediate the acquisition of sulfate at the root surface to initiate this assimilatory metabolic process (Takahashi et al., 2006).
Sulfate uptake and Cys synthesis are activated under low-sulfur conditions where plants require high demands for sulfur metabolites. Up to the present, numerous molecular studies have been performed to characterize the sulfur nutritional responses of metabolic genes in plant sulfur assimilatory pathways. Sulfate transporters adenosine 5'-phosphosulfate reductases and Ser acetyltransferases are the representatives showing significant increase in their mRNAs under sulfur-deficient conditions (Smith et al., 1995
Particularly for the sulfate acquisition process, two high-affinity sulfate transporter genes, SULTR1;1 and SULTR1;2, showed clear responses to sulfur limitation at epidermis and cortex of Arabidopsis roots (Takahashi et al., 2000 Contrary to the cumulative information on transcriptional control, no reports (to our knowledge) have ever demonstrated posttranscriptional control of mRNAs and proteins of sulfate transporters in plants from the perspective of their responses to the changes in environmental sulfur conditions. In this study, we demonstrate that posttranscriptional regulation plays an important role in both sulfur-responsive and organ-specific expression of SULTR1;1 and SULTR1;2 in Arabidopsis roots. These additional regulatory mechanisms can work in parallel with the on-offs of transcription switches for SULTR1;1 and SULTR1;2 gene expression and may have importance in adjustment of sulfate influx under varying sulfur conditions in the environment.
sultr1;1 sultr1;2 Double Knockout Mutant Lacks High-Affinity Sulfate Uptake Activity
The physiological roles of SULTR1;1 and SULTR1;2 high-affinity sulfate transporters were confirmed by analyzing the phenotypes of knockout plants. We isolated transferred DNA (T-DNA) insertion lines (Arabidopsis Knockout Facility at the University of Wisconsin Biotech Center) by PCR-based reverse genetic strategy (Krysan et al., 1999
To analyze the effects of gene disruption of SULTR1;1 and SULTR1;2 on morphological phenotypes, Wassilewskija (Ws) wild-type, 1;1KO, 1;2KO, and DKO plants were grown vertically for 10 d on agar medium containing 1,500 or 5 µM sulfate as the sole sulfur source. The growth of DKO was inhibited under both sulfur-sufficient and -deficient conditions (Fig. 2A ). In particular, the DKO plants were stunted and unable to expand their rosette leaves when grown with 5 µM sulfate (Fig. 2A). Ws and 1;1KO were visibly identical, and their plant biomasses were not significantly different from each other (Fig. 2B). However, 1;2KO plants showed slight growth retardation as compared to Ws, which resulted in a decrease in the plant biomass (Fig. 2B).
The activity of sulfate uptake was analyzed in Ws, 1;1KO, 1;2KO, and DKO plants (Fig. 2C). Plants were grown for 15 d on agar medium containing 1,500 or 5 µM sulfate, and sulfate uptake rates were measured in liquid medium containing 5 µM [35S]-labeled sulfate. DKO plants completely lacked the ability to take up sulfate (Fig. 2C), as expected from their growth defects on low-sulfate medium. The results demonstrate SULTR1;1 and SULTR1;2 are the two essential components of the sulfate uptake system that ultimately facilitates the absorption of sulfate externally supplied to the root system. In contrast to a complete lack of sulfate uptake in DKO under the low-sulfate condition, sulfate transport activities were substantially retained in 1;1KO and 1;2KO plants (Fig. 2C), indicating a single transport component, SULTR1;1 or SULTR1;2, is sufficient to facilitate the uptake of sulfate in Arabidopsis roots. The sulfate uptake rates of 1;2KO plants were 50% to 60% of those of the Ws wild-type plants (Fig. 2C). By contrast, 1;1KO plants showed no significant changes of sulfate uptake activities as compared with Ws plants, irrespective of the sulfur conditions of the preculture (Fig. 2C). However, contribution of SULTR1;1 to the uptake of sulfate was evident from the differences between DKO and 1;2KO.
Using DKO as a parental line, we generated transgenic plants overexpressing epitope-tagged proteins of SULTR1;1 or SULTR1;2 under a constitutive promoter. The c-myc epitope and hexa-His tags were tandemly fused to the end of the coding region of SULTR1;1 and SULTR1;2 (SULTR1;1mycHis and SULTR1;2mycHis, respectively), and the fusion gene constructs were placed under the control of the cauliflower mosaic virus (CaMV) 35S RNA promoter. These fusion constructs were transformed to DKO plants, obtaining four independent, homozygous, single-insertion transgenic lines for each construct. Transgenic lines from both 35S:SULTR1;1mycHis and 35S:SULTR1;2mycHis constructs were able to rescue the growth of DKO parental plants on 5 µM sulfate medium (Fig. 3 ), indicating that both SULTR1;1mycHis and SULTR1;2mycHis can serve as functional sulfate transporters facilitating the uptake of trace amounts of sulfate (5 µM) in the culture medium. In particular, the growth of the aerial part was better improved with SULTR1;2mycHis than with SULTR1;1mycHis (Fig. 3).
SULTR1;1mycHis and SULTR1;2mycHis Transcripts Are Predominantly Found in Roots
The SULTR1;1 and SULTR1;2 mRNA levels were analyzed in 35S:SULTR1;1mycHis and 35S:SULTR1;2mycHis transgenic plants and in Ws wild-type plants grown continuously for 15 d on agar medium containing 1,500 or 5 µM sulfate. The epitope-tagged and endogenous transcripts in transgenic and wild-type plants, respectively, were quantified by real-time reverse transcription (RT)-PCR using gene-specific primers for SULTR1;1 and SULTR1;2. In the roots of wild-type plants, mRNA levels of SULTR1;1 and SULTR1;2 in sulfur-starved plants (5 µM sulfate) were approximately 30 and 9 times higher, respectively, compared to those in the control plants (1,500 µM sulfate; Fig. 4
). These sulfur responses were consistent with the results reported in previous studies (Takahashi et al., 2000
CaMV 35S promoter is suggested to be constitutively active irrespective of plant organs; however, the accumulation of both SULTR1;1mycHis and SULTR1;2mycHis mRNAs occurred predominantly in root tissues (Fig. 4). SULTR1;1mycHis mRNA was detected mainly in the roots of all four 35S:SULTR1;1mycHis transgenic lines, showing 1.5- to 2-fold higher levels of mRNA accumulation in sulfur-starved plants than in the control plants grown with adequate sulfur supply (Fig. 4A). This small increase was statistically significant by paired Student's t test (P = 0.007); however, the rate of induction was rather limited as compared to the strong induction of the endogenous SULTR1;1 mRNA in the wild type. SULTR1;2mycHis mRNA was also regulated posttranscriptionally in 35S:SULTR1;2mycHis plants (Fig. 4B). The SULTR1;2mycHis mRNA was predominantly accumulated in the roots of all four transgenic lines, although the transcript was detected in both roots and leaves (Fig. 4B). As for the sulfur responsiveness, the SULTR1;2mycHis mRNA levels in roots were 2- to 3.5-fold higher in sulfur-starved plants than in the control plants (P = 0.003, paired Student's t test; Fig. 4B). The results indicated that SULTR1;1mycHis and SULTR1;2mycHis mRNAs are strictly regulated to accumulate in roots and are increased by long-term sulfur limitation, despite the fusion constructs were driven by constitutive CaMV 35S promoters.
We further analyzed the effects of sulfur deprivation on protein levels of SULTR1;1mycHis and SULTR1;2mycHis. 35S:SULTR1;1mycHis and 35S:SULTR1;2mycHis transgenic lines were grown for 15 d on agar medium containing 1,500 or 5 µM sulfate, and used for preparation of crude protein extracts and subsequent western-blot analysis. Anti-myc antibody detected a specific band at approximately 59 kD in the extracts from 35S:SULTR1;1mycHis and 35S:SULTR1;2mycHis lines (Fig. 5, A and B ), but not in the extract from Ws wild-type plants (data not shown). These specific bands migrated to somewhat lower positions than the predicted molecular masses of the SULTR1;1mycHis and SULTR1,2mycHis proteins (72.6 and 73.7 kD, respectively). High hydrophobicities of these membrane proteins may have resulted in faster migration on the gel.
Both SULTR1;1mycHis and SULTR1;2mycHis proteins were accumulated significantly in the roots under low-sulfate conditions (Fig. 5). Sulfur starvation caused drastic accumulation of SULTR1;1mycHis protein (Fig. 5A), which cannot be explained by the moderate increase of SULTR1;1mycHis mRNA (Fig. 4A). The results indicated SULTR1;1mycHis is regulated predominantly at the level of protein accumulation upon sulfur limitation. SULTR1;2mycHis protein was also accumulated exclusively in the roots of sulfur-starved plants (Fig. 5B). Although substantial quantities of SULTR1;2mycHis mRNAs were found in leaves as well as in roots of sulfur-starved plants (Fig. 4B), the signals for SULTR1;2mycHis proteins were hardly detectable in the leaves (Fig. 5B). In addition, the results indicated that accumulation of SULTR1;2mycHis protein by sulfur limitation (Fig. 5B) was significantly greater than the increase of SULTR1;2mycHis mRNA in roots (Fig. 4B).
Sulfate uptake rates of transgenic and wild-type plants were measured to determine whether the sulfur-dependent posttranscriptional regulation of SULTR1;1mycHis and SULTR1;2mycHis causes the changes in the capacities of sulfate uptake. Plants were precultured for 15 d under sulfur-sufficient (1,500 µM sulfate) or -deficient (5 µM sulfate) conditions, and the uptake rates were measured in liquid medium containing 5 µM [35S]-labeled sulfate (Fig. 6 ).
When plants were precultured under sulfur-sufficient conditions, 35S:SULTR1;1mycHis transgenic lines showed 1.6 to 2.1 times greater capacities of sulfate uptake compared with the Ws wild-type plants (Fig. 6A, black bars). The sulfate uptake capacities of 35S:SULTR1;1mycHis transgenic lines increased about 7-fold by preculturing the plants under sulfur-deficient conditions. The same low-sulfate culture of Ws plants caused approximately 14-fold increase in sulfate uptake (Fig. 6A). Consequently, the 35S:SULTR1;1mycHis transgenic lines and Ws showed comparable levels of sulfate uptake when plants were precultured under 5 µM sulfate (Fig. 6A). The induction of sulfate uptake by sulfur limitation was fairly consistent with the accumulation of SULTR1;1mycHis proteins in transgenic lines (Fig. 5A); however, the case in Ws wild-type plants may also include the effects of the induction of SULTR1;1 and SULTR1;2 transcripts under the native promoters. The SULTR1;2mycHis overexpressors showed slightly different responses. The sulfate uptake rates in 35S:SULTR1;2mycHis lines were around the same levels as in Ws wild-type plants when plants were precultured under sulfur-sufficient conditions. In three out of four 35S:SULTR1;2mycHis lines, the sulfate uptake rates were elevated by sulfur limitation more significantly than in Ws (Fig. 6B). The induction of sulfate uptake by sulfur limitation was 16- to 24-fold in 35S:SULTR1;2mycHis plants (Fig. 6B), which was higher than in the case of 35S:SULTR1;1mycHis plants (Fig. 6A). The induction of sulfate uptake was generally consistent with the accumulation of SULTR1;2mycHis proteins in roots (Fig. 5B).
To examine the kinetics of the induction of SULTR1;1mycHis and SULTR1;2mycHis, a time-course experiment was performed. Plants were grown for 12 d on sulfur-replete agar medium containing 1,500 µM sulfate, and then transferred onto agar medium containing no sulfur source, harvesting root samples 0, 2, 8, 24, and 72 h after the transfer. The mRNA and protein levels of SULTR1;1mycHis or SULTR1;2mycHis were determined during this time course.
In Ws wild-type plants, SULTR1;1 mRNA increased continuously during the time course of sulfate starvation, resulting in 13-fold induction in 72 h (Fig. 7A
, left). On the other hand, SULTR1;2 mRNA showed a transient increase in the first 2 h but was resumed at 8 h, maintaining its level around 2- to 3-fold of the initial level during the remaining period of sulfur starvation (Fig. 7B, left). Except for this transient induction of SULTR1;2 at 2 h, the accumulation of SULTR1;1 and SULTR1;2 mRNAs during the time course of sulfur limitation from 8 to 72 h was generally consistent with the results of previous reports (Takahashi et al., 2000
In 35S:SULTR1;1mycHis and 35S:SUTLR1;2mycHis transgenic plants, SULTR1;1mycHis and SULTR1;2mycHis transcripts were transiently accumulated (2- to 3-fold), peaking 2 h after the transfer (Fig. 7, A and B, right); however, this temporal induction was not reflected to protein accumulation (Fig. 8, A and B ). During the period from 8 to 72 h, the transcript levels of both SULTR1;1mycHis and SULTR1;2mycHis shifted constantly around the initial level. The results were consistent between the two independent transgenic lines of each construct (Fig. 7, A and B). Contrary to this static behavior of mRNA accumulation during the 8- to 72-h period, the abundance of both SULTR1;1mycHis and SULTR1;2mycHis proteins drastically increased at 8 h and reached the maximum at 24 h after the withdrawal of sulfate (Fig. 8, A and B). It was evident that the accumulation of SULTR1;1mycHis and SULTR1;2mycHis proteins in the overexpressor plants occurs rapidly and effectively by short-term sulfate starvation, although the transgenes were expressed under the constitutive CaMV 35S promoters.
The physiological relevance of protein accumulation was confirmed by measurements of sulfate uptake rates in 35S:SULTR1;1mycHis and 35S:SUTLR1;2mycHis transgenic plants. Transgenic and Ws wild-type plants were precultured for 12 d on agar medium containing 1,500 µM sulfate and subjected to 24-h sulfur starvation. Plants that were not subjected to this short-term sulfur-starvation treatment were used as controls. Both the wild-type and transgenic plants showed nearly the same levels of sulfate uptake under the control condition (Fig. 9 , +S). Under sulfur-starved conditions (Fig. 9, –S), two independent lines of 35S:SULTR1;1mycHis plants, 1;1ox-1 and 1;1ox-2, showed 3.6- and 2.8-fold increase in sulfate uptake rates, respectively. Those were comparable to the induction of sulfate uptake observed in wild-type plants. In 35S:SULTR1;2mycHis plants, the induction of sulfate uptake by 24-h sulfur starvation was significantly higher than in the wild type. The induction of sulfate uptake rates were 6.4- and 5.3-fold for line 1;2ox-11 and line 1;2ox-15, respectively.
In both SULTR1;1mycHis and SULTR1;2mycHis overexpressors, drastic increase of sulfate uptake by sulfur limitation occurred even with constitutive CaMV 35S promoters (Figs. 6 and 9). The induction of sulfate uptake was generally consistent with the increase in protein levels in both long-term (Fig. 5) and short-term (Fig. 8) sulfur-starvation treatments.
Induction of sulfate uptake is the primary response to sulfur-starvation stress. Previously we have reported that SULTR1;1 and SULTR1;2 high-affinity sulfate transporters are expressed at the cortical and epidermal cell layers of Arabidopsis roots (Takahashi et al., 2000 ; Shibagaki et al., 2002; Yoshimoto et al., 2002), and their transcripts are strictly regulated by sulfur status under the control of sulfur-responsive sequences in the promoter regions (Maruyama-Nakashita et al., 2004, 2005). In this report, we proved that SULTR1;1 and SULTR1;2 substantially function as two essential components of the high-affinity sulfate uptake system necessary for the acquisition of sulfate from the environment. We further demonstrated that both SULTR1;1 and SULTR1;2 are controlled posttranscriptionally mainly at the levels of protein accumulation in roots responding to the changes in environmental sulfur conditions. This study provides new regulatory schemes of plant sulfur assimilation in addition to the previously reported transcriptional regulation of the sulfate uptake systems. We first clarified the functional significance of SULTR1;1 and SULTR1;2 for the acquisition of sulfate by analyzing the sulfate uptake capacities of the knockout mutants. DKO plants failed to absorb sulfate from low-sulfate medium (Fig. 2), demonstrating that the high-affinity sulfate transporters SULTR1;1 and SULTR1;2 are essentially required for the uptake of micromolar sulfate from the rhizosphere. In contrast to the severe growth defects and complete loss of sulfate uptake in DKO, both 1;1KO and 1;2KO were viable and retained substantial capacities to take up sulfate from low-sulfate medium (Fig. 2). These results suggested that SULTR1;1 and SULTR1;2 may have overlapping functions and act independently for the uptake of sulfate when either of them is deleted. In fact, overexpression of SULTR1;1mycHis or SULTR1;2mycHis alone conferred sulfate uptake activity in DKO plants (Figs. 3 and 6), suggesting a single transporter component is sufficient for the acquisition of sulfate. Comparison of 1;1KO and 1;2KO suggested that the contribution of SULTR1;2 to total sulfate uptake predominates over the function of SULTR1;1 (Fig. 2C), reflecting the differences of their transcript abundance. Furthermore, we found that overexpression of SULTR1;1mycHis and SULTR1;2mycHis show slight differences in rescuing the sulfate uptake capacities of DKO; the 35S:SULTR1;2mycHis plants were able to absorb sulfate more efficiently compared to the 35S:SULTR1;1mycHis plants during sulfur starvation (Figs. 6 and 9). Consistent with the differences in sulfate uptake rates, recovery of growth was more substantial in 35S:SULTR1;2mycHis than in 35S:SULTR1;1mycHis plants (Fig. 3). It is suggested that SULTR1;2 protein may have a higher capacity for sulfate uptake as compared with SULTR1;1.
The posttranscriptional regulation of SULTR1;1 and SULTR1;2 was determined in transgenic plants overexpressing the epitope-tagged proteins SULTR1;1mycHis and SULTR1;2mycHis in DKO background. Although the epitope-tagged transporters were expressed under the constitutive CaMV 35S promoters, SULTR1;1mycHis and SULTR1;2mycHis mRNAs were predominantly found in roots (Fig. 4). Furthermore, the mRNA levels were slightly increased by a long-term continuous low-sulfur culture (15 d from germination). It was previously reported that the levels of CaMV 35S-driven GFP are unaffected by long-term sulfur limitation (Ohkama et al., 2002 We further performed time-course sulfur-starvation experiments to demonstrate the significance of posttranscriptional regulation of sulfate transporters in short time periods. For the time-course experiment, plants were cultured for 12 d with adequate sulfur supply before transfer to sulfur-less medium. Therefore, the results indicated here represent the comparison of plants at the same developmental stages. SULTR1;1mycHis and SULTR1;2mycHis proteins started to accumulate no later than 8 h after the transfer and reached the maximum levels in 24 h, whereas the levels of their corresponding mRNAs showed no significant increase under the same conditions (Figs. 7 and 8). It is suggested that accumulation of SULTR1;1mycHis and SULTR1;2mycHis proteins during sulfur starvation can be caused by increased translation or by changes in protein turnover/stability. By contrast, accumulation of SULTR1;1mycHis and SULTR1;2mycHis mRNAs occurred under prolonged low-sulfate stress (Fig. 4) or transiently after the replacement of culture medium (Fig. 7, 2 h), the latter of which had no relevance to protein accumulation (Fig. 8). The time-course experiments further indicated that the accumulation of SULTR1;1mycHis or SULTR1;2mycHis proteins in transgenic plants occurs rapidly and even more significantly (Fig. 8) compared with the up-regulation of SULTR1;1 or SULTR1;2 transcripts in Ws wild-type plants (Fig. 7, A and B, left). These results suggest that control of protein level is a dominant mechanism to optimize the uptake of sulfate in response to sulfur deficiency. The mechanism proposed here controls the amounts of SULTR1;1 and SULTR1;2 sulfate transporters and may have close linkage to the regulation of the sulfate uptake process in roots.
The physiological significance of this regulatory mechanism was manifested by the induction of sulfate uptake (Figs. 6 and 9) that occurred in parallel with the increase in SULTR1;1mycHis and SULTR1;2mycHis proteins in sulfur-starved plants (Figs. 5 and 8). The conditions where SULTR1;1mycHis and SULTR1;2mycHis proteins accumulated in overexpressors were identical with those necessary for the promoter-dependent induction of the endogenous SULTR1;1 and SULTR1;2 transcripts (Maruyama-Nakashita et al., 2004
Sulfate transporters contain a conserved region named STAS (sulfate transporter and anti-sigma factor antagonist) domain at their C terminus. The STAS domain shares significant similarity with the Bacillus sp. anti-sigma factor, and is suggested to function as a domain for protein-protein interaction, which may play a role in regulating the activity and/or stability of sulfate transporters. The role of the STAS domain in the control of both activity and biosynthesis/stability of SULTR1;2 has been studied using yeast cells that express SULTR1;2 proteins containing no or mutated STAS domains (Rouached et al., 2005
Besides the regulation of sulfate transporters demonstrated in this study, multiplicity of regulatory mechanisms has been reported for other nutrient uptake systems. The root-specific and iron deficiency-inducible expression of Arabidopsis IRT1, the major transporter for high-affinity iron uptake, was controlled at the levels of both transcription and protein accumulation (Connolly et al., 2002
Plant Materials and Growth Conditions
Arabidopsis (Arabidopsis thaliana) plants were grown on agar medium (Fujiwara et al., 1992
The Arabidopsis sultr1;1 mutant (Wisconsin T-DNA insertion lines, Ws accession, available from the Arabidopsis Biological Resource Center) was isolated by PCR screening as described by Krysan et al. (1999)
Total RNA was extracted using the RNeasy plant mini kit (Qiagen) and treated with DNase I (Invitrogen). RT was carried out using OmniScript reverse transcriptase (Qiagen) and oligo-d(T)12-18. Knockouts of SULTR1;1 and SULTR1;2 (Fig. 1) were confirmed by RT-PCR using the primer pairs described by Yoshimoto et al. (2002)
Real-time RT-PCR was performed for the quantification of SULTR1;1 and SULTR1;2 transcript levels (Figs. 4 and 7). First-strand cDNA was synthesized from 1 µg of total RNA using oligo-d(T)12-18 and OmniScript reverse transcriptase (Qiagen). The first-strand cDNA that derives from 10 ng of total RNA was used for real-time PCR. The reaction was performed using SYBR Premix Ex Taq (Takara), and the signals were detected with 7500 Fast Real-Time PCR system (Applied Biosystems). The SULTR1;1mycHis and SULTR1;2mycHis mRNA levels were determined using the gene-specific primer pairs for SULTR1;1, SULTR1;2, and UBIQUITIN2 (UBQ2), reported previously by Maruyama-Nakashita et al. (2004)
Molecular biological experiments were carried out according to the standard protocols (Sambrook et al., 1989
The 35S:SULTR1;2mycHis fusion was constructed as follows. CaMV 35S promoter fragment from pTH2 vector (Chiu et al., 1996 The kanamycin selection marker in pBI101 (CLONTECH) was replaced with the hygromycin resistance gene. The NheI-HindIII fragment covering the 3'-end region of nopaline synthase promoter, hygromycin phosphotransferase coding region, and polyadenylation signal of Arabidopsis RbcS-2B gene was cut out from the pSMAH vector (provided by Dr. H. Ichikawa, National Institute of Agrobiological Sciences, Japan), and inserted between the NheI and HindIII sites in pBI101 (CLONTECH) for reconstruction of nopaline synthase promoter at the NheI site and for complete replacement of neomycin phosphotransferase II and nopaline synthase terminator with hygromycin phosphotransferase and RbcS-2B terminator. This hygromycin-resistant binary vector was used for plant transformation. The HindIII-SacI fragments of the 35S:SULTR1;1mycHis and 35S:SULTR1;2mycHis fusion cassettes were inserted between the HindIII and SacI sites of this binary vector, replacing the GUS gene.
The resulting binary plasmids were transferred to Agrobacterium tumefaciens GV3101(pMP90) (Koncz and Schell, 1986
Total protein was prepared from the roots and leaves of plants grown vertically on agar medium containing 1,500 or 5 µM sulfate as the sole sulfur source. Tissues were ground under liquid N2 and homogenized in the extraction buffer [50 mM Tris-MES, pH 7.5, 300 mM Suc, 150 mM NaCl, 10 mM CH3COOK, 5 mM EDTA, 20 mM leupeptin, 10 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 200 mM phenylmethylsulfonyl fluoride]. After centrifugation at 9,000g for 10 min to remove cell debris, the supernatant was used as a total protein fraction. Protein content was determined by the Coomassie Blue dye-binding method (Bio-Rad) using bovine serum albumin as a standard.
To detect SULTR1;1mycHis and SULTR1;2mycHis proteins, total protein was mixed with the SDS-PAGE sample buffer and incubated for 30 min at 4°C, and applied to the 7.5% (w/v) polyacrylamide gel. The amount of total protein used for the analysis is described in the figure legends. Protein was separated in the gel and transferred to Hybond-P (Amersham Biosciences). The blot was incubated with anti-myc mouse monoclonal antibody (Invitrogen), followed by the incubation with goat anti-mouse IgG conjugated to alkaline phosphatase (Promega). As a control for equal loading, two different isozymes of Gln synthetase, GS1 and GS2, were detected using anti-GS1 polyclonal antibody and goat anti-rabbit IgG alkaline phosphatase conjugate (Promega) as described previously by Ishiyama et al. (2004)
Experiments were carried out according to the method described by Kataoka et al. (2004b) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AB018695 (SULTR1;1), AB042322 (SULTR1;2), and J05508 (UBQ2).
We thank the Arabidopsis Biological Resource Center and the Arabidopsis Knockout Facility of the University of Wisconsin Biotech Center for providing T-DNA insertion mutants. We thank Dr. Hiroaki Ichikawa (National Institute of Agrobiological Sciences, Japan) for providing the pSMAH vector, and Dr. Yasuo Niwa (University of Shizuoka, Japan) for providing the pTH2 vector. We also thank Dr. Soichi Kojima (RIKEN Plant Science Center) for useful discussions on statistical analysis. Received July 18, 2007; accepted August 22, 2007; published August 24, 2007.
1 This work was supported in part by Special Postdoctoral Fellowship of RIKEN (to N.Y.) and Grants-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H.T.). 
2 Present address: Graduate School of Pharmaceutical Sciences, Chiba University, 1–33 Yayoi-cho, Inage-ku, Chiba 263–8522, Japan. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hideki Takahashi (hideki{at}riken.jp).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.105742 * Corresponding author; e-mail hideki{at}riken.jp.
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