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First published online October 1, 2004; 10.1104/pp.104.047068 Plant Physiology 136:3209-3222 (2004) © 2004 American Society of Plant Biologists Isolation and Characterization of an Arabidopsis Mutant That Overaccumulates O-Acetyl-L-Ser1,[w]Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060–8589, Japan (N.O.-O., S.N.); Department of Applied Biological Chemistry, Graduate School of Agricultural Life Sciences (I.K.), and Biotechnology Research Center (T.F.), The University of Tokyo, Tokyo 113–8657; and Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Saitama 332–0012, Japan (T.F.)
O-Acetyl-L-Ser (OAS) is a positive regulator for the expression of sulfur (S) deficiency-inducible genes. In this study, through the isolation and analysis of Arabidopsis mutants exhibiting altered expression of S-responsive genes, we identified a thiol reductase as a regulator of the OAS levels. Ethyl methanesulfonate-mutagenized M2 seeds of transgenic Arabidopsis NOB7 carrying a chimeric S-responsive promoter driving the green fluorescent protein gene were screened for mutants with altered levels of green fluorescence compared to parental NOB7 line. One of the lines exhibited elevated levels of green fluorescence and mRNA accumulation of several endogenous S-responsive genes and carried a single recessive mutation responsible for the phenotype. OAS concentration in the rosette leaves of the mutant was about five times higher than that of wild-type plants. Based upon the high OAS levels, the mutant was named osh1-1 (OAS high accumulation). The OSH1 locus was mapped to a 30-kb region in chromosome V. DNA sequence analysis revealed no base change in this region; however, a demethylated C residue was found in the first exon of At5g01580. At5g01580 mRNA accumulation was higher in osh1-1 than in wild type, while transcript levels of other genes in the mapped region were not significantly altered in osh1-1. A line of transgenic plants overexpressing At5g01580 had elevated levels of endogenous S-responsive genes. These results suggest that elevated expression of At5g01580 is the cause of osh1 phenotype. Based on sequence similarity to animal thiol reductases, At5g01580 was tested for and exhibited thiol reductase activity. Possible roles of a thiol reductase in OAS metabolism are discussed.
Sulfur (S) is one of the essential elements of higher plants and is an important constituent of proteins, lipids, secondary metabolites, and coenzymes. In proteins, S participates in the reversible formation of disulfide bonds. The importance of S can be further evidenced by the integral role of iron-S proteins in electron transfer in the chloroplast (for review, see Marschner, 1995  ). In addition, glutathione (GSH) has roles as a signaling molecule, is involved in the cell cycle, serves as a source of reducing equivalents in the antioxidative pathway and, by formation of phytochelatins, is involved in the detoxification of heavy metals (for review, see May et al., 1998; Cobbett and Goldsbrough, 2002).
The S metabolic pathway in plants was recently completely defined, and most of the genes encoding enzymes in this pathway have been cloned. Some of enzymes involved in the pathway were activated under S deficiency, and this corresponded to increases in mRNA accumulation (for review, see Leustek et al., 2000
In addition to levels of available S, nitrogen (N) nutrition also regulates S metabolism. Activities of ATP sulfurylase (ATPS) and adenosine 5'-phosphosulfate reductase (APR) were decreased under nitrate deficiency and increased by application of
GSH, a tripeptide of
Aside from the signal metabolites OAS and GSH, little is known about regulation of S metabolism. It is of crucial importance to identify regulatory proteins, if any, involved in regulation of genes for S metabolism. It is also important to reveal the mechanisms of signal molecule homeostasis and of regulation of S metabolism by these signals. To this end, we generated transgenic Arabidopsis plants in which regulation of gene expression by S nutrition was able to be visualized (Ohkama et al., 2002
To gain more insight into the regulation of S metabolism, we exploited a genetic strategy to isolate mutants exhibiting altered response to S nutrition. Response of
Isolation of an S-Response Mutant, osh1-1
Transgenic Arabidopsis NOB7 plants carry GFP reporter gene under the control of the 35S:: In backcrossing experiments with NOB7, the phenotype of all (40/40) F1 plants was wild type when osh1-1 was used as female. When osh1-1 was used as a male, 37 out of 40 F1 plants exhibited wild-type phenotype. The reason why the other three plants were scored as mutants is likely to be due to variation in GFP fluorescence. The phenotype of F2 progeny segregated in a 3:1 ratio (wild type:mutant), indicating that the phenotype is caused by a single nuclear locus (Table I). These findings indicated that the high GFP fluorescence phenotype of osh1-1 was caused by a single recessive mutation.
osh1-1 Shows Higher Expression of S-Responsive Genes osh1-1 was backcrossed three times with the wild-type transgenic NOB7 line, and a line segregating with the osh1 phenotype was established. Wild-type and osh1-1 plants were grown on +S agar plates for 10 d and then transferred to fresh +S or –S agar plates and grown for an additional 4 d. As shown in Figure 1, A and B, GFP fluorescence of osh1-1 was higher than that of wild-type plants under both +S and –S conditions. osh1-1 plants were smaller than wild type with both shoot fresh weight (FW) and root length about one-half as those of wild type (Fig. 1C).
In addition to an increased GFP fluorescence driven by the 35S::
osh1-1 Has Higher OAS, Ser, and Thr Concentrations
Concentration of OAS, a positive regulatory metabolite for S-responsive genes (Kim et al., 1999
Ser Application to Plants Elevates OAS and Thr Levels
As Ser is a precursor of OAS, it was possible that an elevated level of Ser in osh1-1 was a factor in the increase of OAS concentration. To test this hypothesis, we measured OAS concentration in leaves of plants applied with Ser. Wild-type NOB7 plants grown on normal (+S) agar plates for 10 d were transferred to fresh agar plates supplemented with 10, 30, or 100 mM of Ser. Four days following transfer, Ser and OAS concentrations in leaves were determined. Concentration of Ser increased more than 50-fold when Ser was applied at above 10 mM (data not shown). OAS concentration was about 7-, 11-, and 19-fold higher when Ser was applied at 10, 30, and 100 mM, respectively (Fig. 3A), showing the dependence of OAS concentration on the availability of Ser. As the Thr level was increased in osh1-1 under +S condition (Table II), we tested the possibility that an increase in Thr concentration was caused by an increase in Ser concentration. As shown in Figure 3B, Thr level in leaves was increased by Ser application. Level of GFP fluorescence in leaves of wild-type NOB7 plants applied with Ser was also determined (Fig. 3C). GFP fluorescence was significantly increased when Ser was applied at above 30 mM. Application of 100 mM mannitol did not affect levels of GFP fluorescence, indicating that elevation of GFP fluorescence by Ser was not due to an increase in osmotic pressure. In NOC2 plants carrying GFP gene driven under an authentic CaMV 35S promoter (Ohkama et al., 2002
Positional Identification of OSH1 To identify the gene responsible for the mutant phenotype, the osh1 locus was genetically mapped. The mutant (Columbia ecotype [Col-0] accession) was crossed with Landsberg erecta ecotype accession. The F1 plants were selfed and homozygous osh1 plants were selected from the segregating F2 population based on strong GFP fluorescence. After a rough mapping, osh1 was mapped to the top region of Chromosome V. Several new markers were developed based on the genomic sequences of bacterial artificial chromosome (BAC) clones in this region (Supplemental Tables I and II, available at www.plantphysiol.org). Fine mapping using these new markers delimited OSH1 to an approximately 30-kb region of the BAC clone F7A7 between the markers F7A7.2 and F7A7.3 (Fig. 4A). Genomic sequences of wild type and osh1-1 were compared in this region, but no difference was observed.
As we were confident of our mapping data, we examined the possibility that epigenetic differences such as a difference in methylation may be present in this region. To test this, isoschizomeric restriction enzymes HpaII and MspI were used. When the second C of the recognition sequence, CCGG, is methylated, DNA is not cleaved with HpaII while cleaved with MspI. Four overlapping probes covering the mapped region (Fig. 4B) were used to detect the difference in digestion pattern between wild type and osh1-1 by Southern hybridization. When cleaved with HpaII, a 2.3-kb band corresponding to 221,355 to 223,688-bp region of chromosome V was observed when probe "a" was used with genomic DNA from osh1-1, but not with that from wild type. No other difference was observed in the mapped region. The 2.3-kb band was observed with DNA from both genotypes when cleaved with MspI (data not shown). To confirm the result of Southern hybridization, HpaII- or MspI-digested DNA was amplified with two primer sets covering each end of the 2.3-kb fragment (Fig. 4C). With the primers covering the left end, HpaII-digested DNA from both genotypes was not amplified, indicating that DNA was cleaved with HpaII in both genotypes. With primers covering the right end, HpaII-digested DNA from wild type was amplified but not efficiently amplified when DNA from osh1-1 was used. Essentially no amplification was observed with MspI-digested DNA for both ends in both genotypes. These results suggested that the second C of the recognition sequence at 223,689 of chromosome V was methylated in wild type but demethylated in osh1-1.
To directly determine the methylation status, we applied the bisulfite sequencing method (Jacobsen et al., 2000
The C residue at 223,689 of chromosome V is located in the predicted first exon of At5g01580 based on the Arabidopsis Information Resource database (http://www.arabidopsis.org/; Fig. 4, D and E). By using the database information, we isolated cDNA for At5g01580. Sequence of the cDNA was consistent with the exon/intron structure of the translated region in the database. To examine effect of demethylation on mRNA accumulation, real-time PCR analysis was carried out. Relative abundance of At5g01580 mRNAs to ubiquitin5 was about 2-fold higher (P < 0.02 by Student's t test) in osh1-1 than in wild-type NOB7 both under +S and –S conditions (Fig. 5A). Accumulation of At5g01580 mRNAs was not increased by –S in both wild type and osh1-1 (Fig. 5A). Similar trends were also observed when
For further confirmation, a transgenic Arabidopsis line carrying At5g01580 open reading frame (ORF) driven under the CaMV 35S RNA promoter (35S::At5g01580) was obtained. In this line, relative mRNA accumulation of At5g01580 increased about 2,000-fold compared to that in the vector control line (Table IV). Relative mRNA levels of both of APR1 and Sultr2;2 were severalfold higher than those in the vector control line under the +S condition (Table IV). Under the –S condition, accumulation of APR1 and Sultr2;2 mRNAs were not significantly different between the 35S::At5g01580 line and the vector control line, but it tend to be higher in the 35S::At5g01580 line. Although it is absolutely necessary to obtain additional independent transgenic lines to confirm that overexpression of At5g01580 causes elevated accumulation of OAS and mRNAs of APR1 and Sultr2;2, the phenotype of the single transgenic line obtained thus far strongly supports that At5g01580 is the causal gene for the phenotype of osh1-1.
As no expressed sequence tag corresponding to At5g01580 was found in GenBank database, expression of At5g01580 was considered to be very low. In fact, the mRNA level of At5g01580 was about 102- and 104-fold lower than those of ubiquitin5 and  -tubulin, respectively, judging from the amplification curve in real-time PCR analysis (data not shown). To test tissue-specific expression, relative mRNA accumulation of At5g01580 was examined in various tissues by real-time PCR analysis (Fig. 5B). In 2-week-old plants, the levels of At5g01580 mRNAs were about 7-fold higher in roots than in rosette leaves. In rosette leaves, there was no major difference in the mRNA accumulation between 2-week-old and 5-week-old plants. In 5-week-old plants, accumulation of At5g01580 mRNAs in stems and flowers were about 17- and 4-fold higher than those in rosette leaves, respectively.
The predicted protein sequence of At5g01580 (233 amino acids; GenBank accession no. NP_195778) contained the domain of
GILT of human was shown to reduce disulfide bonds optimally at acidic conditions (Arunachalam et al. 2000 ). This protein contains 261 amino acid residues comprised of a 37 aa-long signal peptide and a 22- amino acid-long proform. Both N- and C-termini of the proform are cleaved to generate the mature form of GILT. The proform was shown to localize to the secretory pathway, while the mature form was localized to lysosomes and endosomes. GILT in animals is considered to function in reducing disulfide bonds of antigens in antigen presenting cells, which facilitates subsequent proteolysis (Maric et al., 2001).
Figure 6B shows comparison of the predicted amino acid sequences of At5g01580 and the GILT of human. The predicted sequence of At5g01580 shared relatively high identities and similarities with the human GILT not only for the mature protein region but also for the signal peptide and the C-terminal propeptide regions of human GILT. TargetP (Emanuelsson et al., 2000
As the predicted protein sequence of the At5g01580 shared relatively high identities with GILT from animals, we examined whether At5g01580 protein has thiol reductase activity. Escherichia coli-expressed At5g01580 protein was reacted with cy3-conjugated F(ab')2 fragment of IgG as a substrate, and reduction of F(ab')2 into H' and L chains via Fab' was analyzed by nonreducing SDS-PAGE followed by detection of cy3 fluorescence. As shown in Figure 7, when thioredoxin (positive control) or At5g01580 protein was used, the amounts of F(ab')2 were decreased and signals corresponding to H' and L chains increased at pH 5.0 to 7.0. On the other hand, when lysozyme (negative control) was used, only weak signals of H' and L chains were observed. As pointed out by Arunachalam et al. (2000)
At5g01580 Protein Is Localized to Endoplasmic Reticulum To study subcellular localization in living cells, GFP was fused in-frame to the C terminus of At5g01580 protein and placed under the control of a CaMV 35S promoter. The construct was expressed transiently in epidermal cells of Arabidopsis leaves by microprojectile bombardment. As a control, free GFP was also expressed. The GFP fluorescence was analyzed under a confocal laser scanning microscope. As shown in Figure 8A, free GFP predominantly localized in the nucleus and around the plasma membrane, whereas the GFP-tagged At5g01580 localized in the endoplasmic reticulum (ER)-like reticulate structures in addition to the nucleus and around the plasma membrane (Fig. 8A). Transient expression of these constructs in tobacco Bright Yellow-2 cells showed essentially the same pattern of GFP fluorescence to that in Arabidopsis epidermal cells (Fig. 8B). These results suggested that At5g01580 protein was transported through ER.
In this study, an Arabidopsis mutant, osh1-1, was isolated exhibiting elevated expression of a heterologous S-responsive promoter, P35S:: SRx3. We showed that the expression of endogenous S-responsive genes was also up-regulated in osh1-1. During the course of this study, a mutant having a lesion in the sulfate transporter 1;2 gene (Shibagaki et al., 2002; Yoshimoto et al., 2002) was also isolated (N. Ohkama-Ohtsu, Y. Ide, and T. Fujiwara, unpublished data), which supports the validity of our screening strategy. To our knowledge, this is the first identification of mutants that over-accumulate OAS and of a key gene responsible for S-deficiency regulated gene expression in higher plants.
In osh1-1, Ser and OAS concentrations were elevated (Table II). As Ser is a precursor of OAS, we considered that increase of Ser content is the primary cause for the overaccumulation of OAS. In fact, OAS concentration in leaves was elevated by application of Ser to the culture medium (Fig. 3A). OAS is a metabolite at the marginal step of S and N assimilations and its concentration responds to sulfate and nitrate concentrations in the culture medium (Kim et al., 1999
After the fine mapping and sequencing, it was found that no base change was present in the mapped region of osh1-1 genome, suggesting that the genetic lesion in the osh1-1 genome is epigenetic. The osh1-1 mutant was isolated from ethyl methanesulfonate (EMS) mutagenized M2 population. It is not clear how EMS treatment caused epigenetic changes in At5g01580 in the mutant, but our results suggest that EMS can cause epigenetic changes. The facts that the phenotype of the osh1-1 mutant is stable for at least four generations and that the osh1-1 mutation was unambiguously mapped to about the 30-kb region suggest that the epigenetic lesion of the osh1-1 mutant is stable. Epigenetic changes are in many cases variable, but examples of stable epigenetic changes are also reported. For example, several epigenetic mutations induced by ddm1 mutations in Arabidopsis are stably inherited in the succeeding generations (Kakutani et al., 1999 We therefore examined epigenetic changes in the mapped region. Southern-blot analysis using the methylation-sensitive restriction enzyme revealed a hypomethylation(s) in a CCGG sequence in the ORF of At5g01580 in the osh1-1 genome as compared to the wild-type genome. Bisulfite sequencing analysis revealed a small cluster of methylated C residues, all in dinucleotides CG (CpG), within the At5g01580 ORF, and one of them is specifically demetylated in osh1-1 (Fig. 4). Methylation of C residue in CpG is a common feature in C methylation. Based on these findings, we thought it possible that expression of At5g01580 and/or other genes located very close to At5g01580 is altered in the mutant. Accumulation of transcripts for all genes located in the mapped region was determined and At5g01580 was found to be the only gene whose transcript accumulation was significantly increased in osh1-1 (Fig. 5; Table III). Based on the mapping data, we confirmed that the osh1-1 mutation resides among the 12 genes in the mapped region and epigenetic mutations lead to changes in phenotype through changes in gene expression. Thus, it is most likely that osh1 phenotype was due to increase in expression of At5g01580, which is likely to be caused by demethylation of the C residue at 223689 bp of chromosome V. For further confirmation, we obtained a line of transgenic Arabidopsis plants overexpressing At5g01580 and found that accumulation of APR1 and Sultr2;2 transcripts increased in rosette leaves under +S condition (Table IV). We acknowledge that results from a single line of transgenic plants cannot be used to draw conclusions; however, the phenotype of this transgenic line supports At5g01580 being the causual gene of osh1-1 mutant. It is clearly necessary to obtain additional independent transgenic lines and to determine OAS levels in these transgenic lines before drawing a final conclusion.
DNA methylation has long been a factor affecting transcription. A number of examples have been reported in which transcriptional gene silencing is associated with DNA methylation in the promoter region of a gene (Morel et al., 2000
The predicted amino acid sequence of At5g01580 has a high homology with animal GILT (Fig. 6), a lysosomal thiol reductase. We demonstrated that the E. coli-expressed recombinant At5g01580 protein had thiol reductase activity under neutral conditions (Fig. 7). Reversible formation of disulfide bond is known to be one of mechanisms regulating activity of enzymes. Thioredoxin was shown to regulate formation of disulfide bond of enzymes involved in carbon metabolism (Ruelland and Miginiac-Maslow, 1999
Transient expression analysis of GFP-tagged At5g01580 revealed that the protein is located in the ER-like structure (Fig. 8), suggesting that At5g01580 is transported through ER. This is in agreement with the computer predictions of subcellular localization. It is possible that At5g01580 is transported to an organelle whose pH is neutral. It is worth noting that among the several possible mechanisms for the increase in Ser concentrations in osh1-1, change in photorespiration (Douce et al., 2001
Plant Materials and Culture Conditions
In the experiments described here, wild type refers to the unmutagenized transgenic line of Arabidopsis L. Heynh. ecotype Col-0, NOB7 (Ohkama et al., 2002
Surface sterilized seeds were sown on hydroponic culture medium containing 1.5 mM sulfate (+S condition; Hirai et al., 1995
EMS-mutagenized M2 seeds of NOB7 were surface sterilized and sown either on +S or –S plates. After vernalization for 3 d at 4°C in dark, M2 plants were grown for 14 d before determination of GFP fluorescence. Those M2 plants exhibiting altered GFP fluorescence compared to NOB7 were chosen and transferred to soil. M3 seeds were retested for inheritance of the observed phenotype.
For mapping, mutants (Col-0 ecotype) were crossed with Ler plants. Fourteen-day-old F2 seedlings that displayed the mutant phenotype were chosen based on GFP fluorescence. DNA was extracted from shoots of these plants and analyzed for cosegregation with respect to single sequence length polymorphism markers (Bell and Ecker, 1994
DNA sequence of the mapped region was determined as described (Bartlem et al., 2000
GFP fluorescence was determined as described in Niwa et al. (1999)
To detect methylation of C residue by Southern hybridization, genomic DNA was cleaved with MspI or HpaII, size fractionated by agarose gel electrophoresis, and transferred to a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Uppsala). Four DNA fragments corresponding to 212,250 to 221,751, 218,630 to 229,310, 228,388 to 240,488 and 239,353 to 253,183 bp of chromosome V were cleaved from the BAC clone F7A7 (GenBank accession no. AL161946) with SnaBI, NheI, SpeI, and StuI, respectively, and used as probes. Labeling of probes and hybridization were carried out using Gene Images Random-Prime Labeling and Detection system (Amersham Pharmacia Biotech). To detect C methylation by PCR, genomic DNA digested with HpaII or MspI was amplified by PCR for 23 cycles. Primers 5'-CAACGCGCTTTATTTTCATAG-3' and 5'-TACTCGGTTCTCAACTGGTC-3', and 5'-GTTACTTAGCTTCTCCACTG-3' and 5'-CTTTCCTTGTTTAATGAGACAC-3' were used for amplifying 220,778 to 221,912 and 222,839 to 223,914 bp of chromosome V, respectively.
XhoI-digested genomic DNA isolated from wild-type and osh1-1 plants was treated with sodium bisulfite and amplified by PCR as described (Jacobsen et al., 2000
Real-time PCR analysis was carried out as described (Goto and Naito, 2002
To construct pNO5 that carries At5g01580 ORF driven under the CaMV 35S RNA promoter (35S::At5g01580), reverse-transcribed RNA was amplified using two sets of primers: At5g01580 1F (5'-ACGCGTCGACATGGCATCATATCAGAGGCTTTG-3') and At5g01580 1R (5'-CAAAATGCTTCATCACATCAGG-3'), and At5g01580 2F (5'-TTAATCTCATCCATTGATCTCCA-3') and At5g01580 2R (5'-ATAGTTTAGCGGCCGCTTAGTGATTGGAATAGCAAACTTGG-3'). The two amplified DNA fragments were subjected to a second PCR with primers At5g01580 1F and At5tg01580 2R. The amplified DNA was cloned into 35S
To construct pNO7 that carries GFP-tagged At5g01580 ORF, reverse-transcribed RNA was amplified using two sets of primers: At5g01580 1F (5'-ACGCGTCGACATGGCATCATATCAGAGGCTTTG-3') and At5g01580 1R (5'-CAAAATGCTTCATCACATCAGG-3'), and At5g01580 2F (5'-TTAATCTCATCCATTGATCTCCA-3') and At5g01580 2R (5'-CATGCCATGGAACCACCACCACCACCACCGTGATTGGAATAGCAAACTTGGG-3'). The two amplified DNA fragments were subjected to a second PCR with primers At5g01580 1F and At5tg01580 2R, which gave rise to the At5g01580 ORF having six Gly codons in place of the termination codon and upstream of the NcoI site. The DNA was cloned into 35S
Microprojectile bombardment was performed as described in Hagiwara et al. (2003)
To construct the plasmid pNO9 for production of recombinant At5g01580 protein, the At5g01580 ORF was amplified as in pNO7 except that At5g01580 3R primer (5'-CCGCTCGAGGTGATTGGAATAGCAAACTTGGG-3') was used in place of At5g01580 2R. The At5g01580 ORF in the amplified fragment is devoid of the termination codon and is flanked by SalI and XhoI sites. The DNA was cloned into pIVEX2.3-MCS in RTS 500 E. coli circular template kit (Roche Diagnostics, Mannheim, Germany) between SalI and XhoI sites to generate the At5g01580 ORF tagged with six His codons at the C terminus. His-tagged At5g01580 protein was expressed in the RTS 500 instruments (Roche Diagnostics) and purified using Ni-NTA agarose (Qiagen, Valencia, CA) as recommended by the manufacturers.
In vitro assay for thiol reductase activity was performed based on the method of Arunachalam et al. (2000)
We thank the Arabidopsis Biological Resource Center (Columbus, OH) for a BAC clone F7A7. We are grateful to Kumi Fujiwara for general assistance, Miwa Hashimoto, Mao Sugasawa, Reiko Mishina, Yuko Kawara, and Kayoko Aizawa for excellent technical assistance, and to Dr. Annita G. Peterson for careful reading of the manuscript. We used the Radioisotope Laboratory of the Graduate School of Agriculture, Hokkaido University. Received June 1, 2004; returned for revision July 26, 2004; accepted July 26, 2004.
1 This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant no. 12138201 to S.N. and T.F.), by a 21st COE project (to T.F.), and by Research Fellowships of Japan Society for the Promotion of Science (grant no. 9846 to N.O.). 
2 Present address: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA 50011.
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.047068. * Corresponding author; e-mail atorufu{at}mail.ecc.u-tokyo.ac.jp; fax 81–3–5841–2408.
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to the medium in Lemna minor and cultured tobacco (Nicotiana tabacum) cells (Brunold, 1993
-Glu-Cys-Gly, is another molecule regulating S metabolism. GSH is the major form of available S within the plant (May et al., 1998
-sGFP(S65T) plasmid (Chiu et al., 1996
