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Four rice genes encoding cysteine synthase: isolation and differential responses to sulfur, nitrogen and light
Gene 229 (1999) 155–161
Four rice genes encoding cysteine synthase: isolation and differential responses to sulfur, nitrogen and light T. Nakamura, Y. Yamaguchi, H. Sano * Nara Institute of Science and Technology, Research and Education Center for Genetic Information, Ikoma, Nara, Japan Received 2 November 1998; accepted 4 January 1999; Received by H. Uchimiya
Abstract Four cDNA clones, rcs1, rcs2, rcs3 and rcs4, encoding cysteine synthase [O-acetylserine(thiol )lyase] were isolated from rice. The predicted amino acid sequences contain the conserved PXXSVKDR region characteristic of cysteine synthase, which includes the lysine residue that binds the cofactor, pyridoxal 5∞-phosphate. Molecular phylogenic analysis suggests that, whereas rcs1 and rcs3 belong to the cytosolic isoform family, rcs2 and rcs4 form a new family of cysteine synthase. Transcript accumulation of each gene was examined for organ specificity, and also for response to sulfur, nitrogen and light. The rcs1 transcript accumulated in all organs examined, and was induced in shoots and roots upon sulfur starvation under non-limiting nitrogen conditions. The rcs2 transcript accumulated in shoots grown in the light, but disappeared almost completely by dark treatment. The rcs3 transcript was found more abundantly in roots than in shoots, and was reduced in the dark, as well as under sulfur and nitrogen deprivation. The rcs4 transcript was scarce in all organs examined. These observations indicate that cysteine synthase genes encode functionally distinct cysteine synthase isoforms, and that they are coordinately regulated by the availability of sulfur, nitrogen, and light. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Cysteine synthesis; O-acetylserine(thiol )lyase; Oryza sativa; Stress response
1. Introduction Using inorganic sulfate from the soil, plants and bacteria synthesize a variety of sulfur compounds, including sulfur-containing amino acids, coenzymes and sulfolipids. Incorporated sulfate is activated and reduced to sulfide by several enzymes and incorporated into cysteine (Anderson, 1990). The final step of cysteine synthesis is catalyzed by cysteine synthase (CS), or Oacetylserine(thiol )lyase, which transfers sulfide to O-acetylserine and releasing acetate. CS has been purified from several plant species, and found to be classified into distinct isoforms that localize to particular cellular compartments such as, plastids, mitochondria and the cytosol (Lunn et al., 1990; Rolland et al., 1992; Yamaguchi et al., 1998). cDNA clones encoding several CS isoforms have been isolated from higher plants, including spinach (Hell et al., 1993; Saito * Corresponding author. Tel.: +81 743-72-5650; fax: +81 743-72-5659; e-mail: [email protected]. Abbreviations: CS, cysteine synthase; dbEST, database for EST; EST, expressed sequence tag; RACE, rapid amplification of cDNA ends.
et al., 1992, 1993a, 1994), Arabidopsis thaliana (Hell et al., 1994; Barroso et al., 1995; Hesse and Altmann, 1995; Nakamura et al., 1997), Brassica juncea (Schafer et al., 1998), wheat ( Youssefian et al., 1993), watermelon (Noji et al., 1994), bell pepper (Romer et al., 1992), maize (Brander et al., 1995) and potato (Hesse and Hoefgen, 1998). Since more than three CS genes have been isolated from Arabidopsis thaliana, some cellular compartments should contain more than two isoforms of this enzyme. However, the physiological significance of multiple cysteine synthetic pathways in a cellular compartment is not clear. The regulation of cysteine synthesis is further complicated by the fact that CS is regulated not only by sulfur, but also by nitrogen availability (Reuveny et al., 1980; Barroso et al., 1995; Takahashi and Saito, 1996; Takahashi et al., 1996) and light ( Kitamura et al., 1996). Sulfur availability was also shown to affect nitrogen metabolism (Reuveny et al., 1980) and photosynthesis ( Wykoff et al., 1998). These observations suggest, first, a coordinated regulation of CS isoforms in different cellular compartments, and second, cross-talk among sulfur and nitrogen metabolism and photosynthesis.
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However, substantial evidence for such a mechanism is minimal. In order to understand the regulatory mechanism of cysteine synthesis, it is necessary to isolate all CS genes from a given plant. In this paper, we report the isolation of four rice CS genes, rcs1, rcs2, rcs3 and rcs4, whose transcripts differentially accumulate in response to sulfur and nitrogen availability and also to light conditions.
2. Materials and methods 2.1. Plant materials and stress conditions Rice (Oryza sativa cv. Nipponbare) seedlings were grown in the 0.5× diluted medium described by Mae and Ohira (1981) under continuous light for 2 weeks, and then subjected to various stress treatments for 6 days, including sulfur starvation (−S ), nitrogen starvation (−N ), sulfur and nitrogen starvation (−SN ) and darkness (D). The nutrient-deprived media were made by replacing SO2− and NO− salts with Cl− salts. 4 3 2.2. cDNA isolation In order to identify rice EST clones corresponding to CS, amino acid sequences from three spinach CS isoforms, CysA (Saito et al., 1992), CysB (Saito et al., 1993a) and CysC (Saito et al., 1994), which localize to the cytosol, plastid and mitochondria, respectively, were aligned to rice sequences from the dbEST using the TBLASTN program. Four independent EST clones with Accession Nos D47342, D46578, D47659 and D24907 were found, which appeared to encode for CS. The corresponding cDNAs were obtained from the MAFF DNA Bank (Ministry of Agriculture, Forestry and Fisheries of Japan). As some of the cDNAs were found to lack the 5∞-terminal region, 5∞RACE-PCR was performed to obtain full-length cDNAs. DNA sequences were determined with the ABI PRISM@ cycle sequencing kit (Perkin-Elmer, Palo Alto, CA) using automated sequencers (Model 373A and Model 310, Applied Biosystems, Foster City, CA). 2.3. DNA blot analysis Total DNA was prepared from rice leaves using the cetyltrimethylammonium bromide precipitation method (Murray and Thompson, 1980). For DNA blot analysis, 20 mg of DNA were digested with an appropriate restriction enzyme, separated on a 0.8% agarose gel and transferred to a Hybond- N+ nylon membrane (Amersham, Cleveland, OH ). The blot was hybridized with 32P-labeled probe and washed with 0.1× SSC containing 0.5% SDS for 1 h at 65°C.
2.4. RNA blot analysis For organ specificity analysis, mature plants grown in Wagner pots under greenhouse conditions were used for total RNA extraction from roots, upper leaves, middle leaves, lower leaves, upper culm, lower culm and panicles. For stress-response analysis, total RNA was isolated from shoots and roots of 80 seedlings grown for 3 weeks. RNA samples were prepared by the aurin tricarboxylic acid method ( Verwoerd et al., 1989). Total RNA, 10 mg per lane were fractionated on a 0.8% agarose gel, and blotted on to a Hybond-N nylon membrane (Amersham) using an apparatus for slot blotting (MilliBlot-S, Millipore). Hybridization was performed with 32P-labeled DNA fragments of CS clone inserts obtained by digestion with an appropriate restriction enzyme. The blot was hybridized and washed at 65°C in 0.1× SSC containing 0.5% SDS for 1 h. Crosshybridization signals among four isoforms were not detectable under these conditions.
3. Results We identified four cDNAs that had a high similarity to spinach CS sequences and designated these as rcs1, rcs2, rcs3 and rcs4. The molecular size of the cDNAs is 1290 bp for rcs1, 1547 bp for rcs2, 1363 bp for rcs3 and 1310 bp for rcs4. They have open reading frames encoding polypeptides of 321, 357, 325 and 339 amino acids, respectively. Comparison of the deduced amino acid sequences with those of spinach CS suggests that the four rice isoforms are CS genes (Fig. 1). This is confirmed by the presence of a Lys residue, which binds the pyridoxal 5∞-phosphate cofactor (Saito et al., 1993b) and the neighboring residues (PXXSVKDR), which are well conserved among plant CSs (Fig. 1). A molecular phylogenic tree based on the amino-acid sequences of the CSs shows that rcs1 and rcs3 belong to the cytosolic isoform family. Although rcs2 and rcs4 are related to the mitochondrial isoform (cysC family), they are likely to form a new family within the CS superfamily (Fig. 2). A genomic DNA blot analysis was carried out to determine the number of sequences of each isoform. Genomic DNA was digested with BamHI and EcoRI, neither of which has any cleavage sites within the cDNAs used as probes. Genomic DNA was also digested with HindIII, by which rcs1 and rcs3 cDNAs are cut at one site, whereas rcs2 and rcs4 cDNAs are not. The resulting DNAs were then subjected to hybridization analysis with the corresponding cDNA probes (Fig. 3). In addition to single band patterns, additional signals were observed in the BamHI digests of rcs2, in the EcoRI digests of rcs1 and rcs3, and in the HindIII digests of rcs1 and rcs2. This may be due to the presence of cleavage sites within the intron sequences of the genes.
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Fig. 1. Amino-acid alignment of rice CSs. The sequences are rcs1, rcs2, rcs3, rcs4, cytosolic type of Spinacea oleracea (spinach cyt) (Saito et al., 1992), plastidic type (spinach chl ) (Saito et al., 1993a) and mitochondrial type (spinach mit) (Saito et al., 1994). Identical amino-acid residues are shaded. Dashes indicate gaps introduced to optimize the alignment. The asterisks show the conserved region surrounding the pyridoxal 5∞-phosphatebinding Lys residue, which is indicated by the arrow. The sequence data have been deposited in the GenBank, EMBL and DDBJ databases under the Accession Nos AF073695 (rcs1), AF073696 (rcs2), AF073697 (rcs3) and AF073698 (rcs4).
Organ-specific accumulation of transcripts for each isoform in mature plants was examined by RNA blot analysis (Fig. 4). Under the experimental conditions used, cross-hybridization among the four isoforms did not occur (data not shown). The relative levels of the transcripts were estimated by densitometrically quantifying the hybridization intensities, except for rcs4 with an absolutely low transcript level for quantification (Fig. 4B). Transcripts for rcs1 were observed in both shoots and roots, whereas those for rcs2 accumulated only in shoots, and those for rcs3 were found more abundantly in roots than in shoots. The level of rcs4 transcript was very low in all organs tested. The effects of environmental conditions on transcript
accumulation were examined for each isoform (Fig. 5). Seedlings were grown in the 0.5× diluted medium described by Mae and Ohira (1981) for 2 weeks and then grown for 6 days under conditions of sulfur starvation, nitrogen starvation, sulfur and nitrogen starvation, or darkness. Total RNA was isolated from shoots and roots, and the transcript levels of rcs1, rcs2, rcs3 and rcs4 were examined by RNA blot hybridization. The relative levels of the transcripts were estimated by densitometrically quantifying the hybridization intensities, except for rcs4 with an absolutely low transcript level for quantification ( Fig. 5B). Upon sulfur deprivation, the level of rcs1 transcripts was increased in both shoots and roots. However, this response had an absolute
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Fig. 3. DNA blot analysis of rice genomic DNA. Total DNA from rice plants was digested with BamHI ( lane 1), EcoRI ( lane 2) or HindIII ( lane 3). After electrophoretic fractionation and blotting to a nylon membrane, DNA fragments were probed with the corresponding 32P-labeled cDNA. Fig. 2. Phylogenic tree of CSs. The tree was constructed by the neighbor-joining method using CLUSTALW version 1.7 (Thompson et al., 1994) and drawn with TV version 1.5 (Page, 1996). The amino acid sequences are from the rice isoforms (rcs1, rcs2, rcs3, rcs4), Arabidopsis thaliana cytosolic [cytACS1 (Hesse and Altmann, 1995), A.t.OAS.5-8 (Hell et al., 1994)], plastidic [A.t.OAS.7-4 (Hell et al., 1994)], putative mitochondrial (mtACS1, database Accession No. X81973), putative organellar type [CS26 (Nakamura et al., 1997)], Spinacia oleracea (spinach) cytosolic [cysA (Saito et al., 1992)], plastidic [cysB (Saito et al., 1993a)], mitochondrial [cysC (Saito et al., 1994)], Citrullus lanatus (watermelon) cytosolic [cysA (Noji et al., 1994)], Capsicum annuum (bell pepper) chromoplastic [CS C.ann. (Romer et al., 1992)], Triticum aestivum (wheat) cytosolic [cys1 ( Youssefian et al., 1993)], Zea mays (maize) putative cytosolic [Mcysp (Brander et al., 1995)], Solanum tuberosum (potato) cytosolic [StCS-A (Hesse and Hoefgen, 1998)], plastidic [StCS-B (Hesse and Hoefgen, 1998)], Escherichia coli [cysK (Sirko et al., 1990)], and Synechocystis sp. [cysK ( Kaneko et al., 1996)]. The families corresponding to the cysA family, cysB family, cysC family, cs26 family, rcs2 family and bacterial family are indicated by brackets. Numbers along the internodes show the bootstrap probability as a percentage, based on 1000 replicate analyses. The scale indicates the substitutions per site.
requirement for nitrogen, as is clearly shown by the insensitivity to sulfur deprivation in the absence of nitrogen ( Fig. 5B). Also, the response of rcs1 was more sensitive to sulfur deprivation in the roots than in the shoots. Transcript accumulation of rcs3 was generally dependent on light and nutritional deprivation in both shoots and roots. Transcript accumulation of rcs2 was similar to rcs3, except that 6 days of darkness almost completely erased its expression in the shoots. The 109th version of the GenBank database revealed 31 rice EST sequences, of which putative translation products have a high similarity to CSs from plants and bacteria. Twenty-two of these ESTs correspond to the four rice sequences reported here. Nine additional ESTs segregate in four contigs, designated as rcs5, rcs6, rcs7
and rcs8. Each contig was analyzed using the BLASTX program, and the proteins that gave the highest similarity are identified ( Table 1). The Accession Nos for each EST are as follows: rcs1 (D47623, D41715, D47342, D22475, C72939, D41186, D41333, D41092, D41319, D22447, D22394, C97464), rcs2 (D46578, D42350), rcs3 (D47659, C71750, C99640, AU032674, C98455, C99702), rcs4 (D24907, AU031942), rcs5 (C74831, D10957, D48020, D21271), rcs6 (D24896), rcs7 (D23867), rcs8 (C26145, D47368, D47728).
4. Discussion Cysteine synthases of higher plants have been found to constitute several isoforms, which localize to different subcellular compartments. To our knowledge, however, no report has so far been presented on CSs from rice. To investigate the physiological role of rice CS isoforms, we have isolated four cDNAs, rcs1, rcs2, rcs3 and rcs4, that encode CSs. Three of these, rcs1, rcs3 and rcs4, lack an extended N-terminal amino acid sequence ( Fig. 1). This would indicate that rcs1, rcs3 and rcs4 localize to the cytosol, although rcs4, together with rcs2, forms a new CS family. The rcs2 has a putative transit peptide sequence at the N-terminus, but it does not show any obvious features of transit peptides for any organelle. The phylogenic tree ( Fig. 2) indicates that the CSs identified so far are divided into six families, which consist of the cysA family (cytosolic isoform), cysB family (plastidic isoform), cysC family (mitochondrial isoform), cs26 family, rcs2 family, and bacterial family.
T. Nakamura et al. / Gene 229 (1999) 155–161
Fig. 4. Organ specificity of transcript accumulation. (A) Total RNA isolated from roots, upper leaves, middle leaves, lower leaves, upper culm, lower culm and panicles of mature rice was fractionated by agarose gel electrophoresis, blotted on to a nylon membrane, and hybridized with 32P-labeled probes. L, lower parts; M, middle parts; U, upper parts. (B) The relative levels of the transcripts were estimated by densitometrically quantifying the hybridization intensities in comparison with those from panicles (100%), except for rcs4 with an absolutely low transcript level for quantification. The intensity of each band was normalized with those of actin bands.
The rcs1 transcript levels were increased within 1 day of sulfur starvation, reaching 1.5- to twofold higher levels after 6 days in comparison with the controls. This response was more pronounced in roots than in shoots. Elevated transcript accumulation in response to sulfur starvation was also observed in other genes involved in the sulfur assimilation pathway, e.g. genes encoding the sulfate transporter (Smith et al., 1995, 1997; Takahashi et al., 1997), ATP sulfurylase (Logan et al., 1996) and APS reductase (Gutierrez-Marcos et al., 1996). This suggests that effective synthesis of cysteine under sulfurdeficient conditions requires the transcriptional
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Fig. 5. Response of transcript accumulation to sulfur, nitrogen and light. Total RNA was isolated from shoots (S ) and roots (R) of plants grown under conditions with full nutrient (C ), sulfur starvation (−S ), nitrogen starvation (−N ), sulfur and nitrogen starvation (−SN ) and darkness (D). Rice seedlings were grown in the 0.5× diluted medium described by Mae and Ohira (1981) at 30°C under continuous light for 2 weeks and subsequently transferred to the indicated condition for 1 week. (A) Total RNA (10 mg) was slot-blotted onto a nylon membrane (Hybond N, Amersham) with a slot-blot apparatus (Millipore), and then hybridized with 32P-labeled probes. (B) Relative mRNA abundance was determined from hybridization intensities of the bands using the BAS-2000 image analyzer (Fuji). The background value, which was calculated from the area just below each bands, was subtracted from the band intensity. Relative mRNA levels are shown in comparison with those from the control conditions of shoots (100%).
induction of a particular CS isoform together with other genes involved in the cysteine synthetic pathway. The induction of rcs1 transcripts by sulfur starvation requires nitrogen to be present in the culture medium.
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Table 1 BLASTX analysis results for contigs from rice ESTs corresponding to cysteine synthase genes Contiga
Number of ESTsb
Similar toc
rcs5 rcs6 rcs7 rcs8
4 1 1 3
Cysteine Cysteine Cysteine Cysteine
synthase synthase synthase synthase
chloroplast precursor chloroplast precursor mitochondrial precursor mitochondrial precursor
Protein IDd
Identity (%)e
Overlapf
P32260 P31300 A55450 A55450
81 82 83 48
215 34 113 89
a Name of the contig (one for each different sequence). b Number of ESTs used to construct each contig. c For which the best similarity was found. d PIR or SwissProt ID of the protein e Percentage of identical residues in the alignment. f Number of amino-acid residues overlapping between a query sequence and its matched protain sequence.
This implies that, in addition to sulfur, rcs1 gene expression is coordinately controlled by nitrogen nutrition, which is necessary to provide one of the substrates for CS, O-acetylserine. Conceivably, rcs1 is involved in the regulatory system that controls the general synthetic balance between sulfur-containing amino acids and other amino acids. The rcs2 transcripts accumulated in green tissues under light, but disappeared completely under dark conditions, indicating that expression of rcs2 is lightdependent. In spinach, synthesis of plastidic CS was induced by light ( Kitamura et al., 1996). Our results together, with these observations, suggest that transcript levels of the rcs2 type CS are concomitantly regulated by photosynthesis, which provides the ATP and reduced ferredoxin necessary for the activation and reduction of sulfate. In addition to an energy supply, photosynthesis also provides carbohydrates with which the carbon skeletons of all amino acids are constructed. The rcs3 transcripts are more abundant in roots than shoots and are reduced under dark conditions, indicating that rcs3 is correlated with photosynthesis in a way similar to that of rcs2. As rcs3 transcript levels were clearly reduced by sulfur and nitrogen deprivation, nutrition and energy possibly regulate its expression in general. The transcript level of rcs4 was very low under all conditions in all organs examined. The expression of rcs4, if any, may be limited to specific cell types and/or environmental conditions. Recently, it has been reported for cocklebur seeds that all cytosolic b-cyanoalanine synthase activity is due to the three cytosolic isoforms of CS (Maruyama et al., 1998). b-cyanoalanine synthase catalyzes the synthesis of b-cyanoalanine from cysteine and cyanide. This implies that multiple CS isoforms are necessary in order to coordinate cysteine and b-cyanoalanine metabolism, although the nature of b-cyanoalanine metabolism is currently unclear. Analyzing rice EST sequences, we identified nine more sequences that showed a high degree of similarity to plant CS ( Table 1). They were grouped into four
contigs, which were designated as rcs5, rcs6, rcs7 and rcs8. The deduced amino-acid sequences from contigs rcs5 and rcs6 revealed a high similarity to plastidic CS, and those from rcs7 and rcs8 had a greater similarity to mitochondrial CS. Contigs rcs5 and rcs7 encode the N-terminal regions, and rcs6 and rcs8 the C-terminal regions of CS, leaving the possibility that they overlap each other. These results indicate that rice contains two to four extra CS genes, in addition to our four genes. However, the exact number of CS isoforms present in any plant is currently not clear. In conclusion, the expression of genes involved in the sulfur assimilation pathway may also depend on the presence of nutrients other than sulfur, nutrient ratios, as well as on light. Further experiments are necessary to understand sulfur metabolism by investigating the spatial gene expression of all CS isoforms under various nutritional conditions.
Acknowledgements The authors thank Drs R. Winz (Max Planck Institute) and T. Kusano (Nara Institute of Science and Technology) for critical reading of the manuscript and suggestion, and the MAFF DNA Bank of National Institute of Agrobiological Resources at Ministry of Agriculture, Forestry and Fisheries of Japan, for the EST clones, D47342, D46578, D47659 and D24907. This work was partly supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture, Japan (06271266) and a grant for Research for the Future Program (JSPS-RFTF 1997R16001) from the Japan Society for the Promotion of Science.
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Four rice genes encoding cysteine synthase: isolation and differential responses to sulfur, nitrogen and light T. Nakamura, Y. Yamaguchi, H. Sano * Nara Institute of Science and Technology, Research and Education Center for Genetic Information, Ikoma, Nara, Japan Received 2 November 1998; accepted 4 January 1999; Received by H. Uchimiya
Abstract Four cDNA clones, rcs1, rcs2, rcs3 and rcs4, encoding cysteine synthase [O-acetylserine(thiol )lyase] were isolated from rice. The predicted amino acid sequences contain the conserved PXXSVKDR region characteristic of cysteine synthase, which includes the lysine residue that binds the cofactor, pyridoxal 5∞-phosphate. Molecular phylogenic analysis suggests that, whereas rcs1 and rcs3 belong to the cytosolic isoform family, rcs2 and rcs4 form a new family of cysteine synthase. Transcript accumulation of each gene was examined for organ specificity, and also for response to sulfur, nitrogen and light. The rcs1 transcript accumulated in all organs examined, and was induced in shoots and roots upon sulfur starvation under non-limiting nitrogen conditions. The rcs2 transcript accumulated in shoots grown in the light, but disappeared almost completely by dark treatment. The rcs3 transcript was found more abundantly in roots than in shoots, and was reduced in the dark, as well as under sulfur and nitrogen deprivation. The rcs4 transcript was scarce in all organs examined. These observations indicate that cysteine synthase genes encode functionally distinct cysteine synthase isoforms, and that they are coordinately regulated by the availability of sulfur, nitrogen, and light. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Cysteine synthesis; O-acetylserine(thiol )lyase; Oryza sativa; Stress response
1. Introduction Using inorganic sulfate from the soil, plants and bacteria synthesize a variety of sulfur compounds, including sulfur-containing amino acids, coenzymes and sulfolipids. Incorporated sulfate is activated and reduced to sulfide by several enzymes and incorporated into cysteine (Anderson, 1990). The final step of cysteine synthesis is catalyzed by cysteine synthase (CS), or Oacetylserine(thiol )lyase, which transfers sulfide to O-acetylserine and releasing acetate. CS has been purified from several plant species, and found to be classified into distinct isoforms that localize to particular cellular compartments such as, plastids, mitochondria and the cytosol (Lunn et al., 1990; Rolland et al., 1992; Yamaguchi et al., 1998). cDNA clones encoding several CS isoforms have been isolated from higher plants, including spinach (Hell et al., 1993; Saito * Corresponding author. Tel.: +81 743-72-5650; fax: +81 743-72-5659; e-mail: [email protected]. Abbreviations: CS, cysteine synthase; dbEST, database for EST; EST, expressed sequence tag; RACE, rapid amplification of cDNA ends.
et al., 1992, 1993a, 1994), Arabidopsis thaliana (Hell et al., 1994; Barroso et al., 1995; Hesse and Altmann, 1995; Nakamura et al., 1997), Brassica juncea (Schafer et al., 1998), wheat ( Youssefian et al., 1993), watermelon (Noji et al., 1994), bell pepper (Romer et al., 1992), maize (Brander et al., 1995) and potato (Hesse and Hoefgen, 1998). Since more than three CS genes have been isolated from Arabidopsis thaliana, some cellular compartments should contain more than two isoforms of this enzyme. However, the physiological significance of multiple cysteine synthetic pathways in a cellular compartment is not clear. The regulation of cysteine synthesis is further complicated by the fact that CS is regulated not only by sulfur, but also by nitrogen availability (Reuveny et al., 1980; Barroso et al., 1995; Takahashi and Saito, 1996; Takahashi et al., 1996) and light ( Kitamura et al., 1996). Sulfur availability was also shown to affect nitrogen metabolism (Reuveny et al., 1980) and photosynthesis ( Wykoff et al., 1998). These observations suggest, first, a coordinated regulation of CS isoforms in different cellular compartments, and second, cross-talk among sulfur and nitrogen metabolism and photosynthesis.
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However, substantial evidence for such a mechanism is minimal. In order to understand the regulatory mechanism of cysteine synthesis, it is necessary to isolate all CS genes from a given plant. In this paper, we report the isolation of four rice CS genes, rcs1, rcs2, rcs3 and rcs4, whose transcripts differentially accumulate in response to sulfur and nitrogen availability and also to light conditions.
2. Materials and methods 2.1. Plant materials and stress conditions Rice (Oryza sativa cv. Nipponbare) seedlings were grown in the 0.5× diluted medium described by Mae and Ohira (1981) under continuous light for 2 weeks, and then subjected to various stress treatments for 6 days, including sulfur starvation (−S ), nitrogen starvation (−N ), sulfur and nitrogen starvation (−SN ) and darkness (D). The nutrient-deprived media were made by replacing SO2− and NO− salts with Cl− salts. 4 3 2.2. cDNA isolation In order to identify rice EST clones corresponding to CS, amino acid sequences from three spinach CS isoforms, CysA (Saito et al., 1992), CysB (Saito et al., 1993a) and CysC (Saito et al., 1994), which localize to the cytosol, plastid and mitochondria, respectively, were aligned to rice sequences from the dbEST using the TBLASTN program. Four independent EST clones with Accession Nos D47342, D46578, D47659 and D24907 were found, which appeared to encode for CS. The corresponding cDNAs were obtained from the MAFF DNA Bank (Ministry of Agriculture, Forestry and Fisheries of Japan). As some of the cDNAs were found to lack the 5∞-terminal region, 5∞RACE-PCR was performed to obtain full-length cDNAs. DNA sequences were determined with the ABI PRISM@ cycle sequencing kit (Perkin-Elmer, Palo Alto, CA) using automated sequencers (Model 373A and Model 310, Applied Biosystems, Foster City, CA). 2.3. DNA blot analysis Total DNA was prepared from rice leaves using the cetyltrimethylammonium bromide precipitation method (Murray and Thompson, 1980). For DNA blot analysis, 20 mg of DNA were digested with an appropriate restriction enzyme, separated on a 0.8% agarose gel and transferred to a Hybond- N+ nylon membrane (Amersham, Cleveland, OH ). The blot was hybridized with 32P-labeled probe and washed with 0.1× SSC containing 0.5% SDS for 1 h at 65°C.
2.4. RNA blot analysis For organ specificity analysis, mature plants grown in Wagner pots under greenhouse conditions were used for total RNA extraction from roots, upper leaves, middle leaves, lower leaves, upper culm, lower culm and panicles. For stress-response analysis, total RNA was isolated from shoots and roots of 80 seedlings grown for 3 weeks. RNA samples were prepared by the aurin tricarboxylic acid method ( Verwoerd et al., 1989). Total RNA, 10 mg per lane were fractionated on a 0.8% agarose gel, and blotted on to a Hybond-N nylon membrane (Amersham) using an apparatus for slot blotting (MilliBlot-S, Millipore). Hybridization was performed with 32P-labeled DNA fragments of CS clone inserts obtained by digestion with an appropriate restriction enzyme. The blot was hybridized and washed at 65°C in 0.1× SSC containing 0.5% SDS for 1 h. Crosshybridization signals among four isoforms were not detectable under these conditions.
3. Results We identified four cDNAs that had a high similarity to spinach CS sequences and designated these as rcs1, rcs2, rcs3 and rcs4. The molecular size of the cDNAs is 1290 bp for rcs1, 1547 bp for rcs2, 1363 bp for rcs3 and 1310 bp for rcs4. They have open reading frames encoding polypeptides of 321, 357, 325 and 339 amino acids, respectively. Comparison of the deduced amino acid sequences with those of spinach CS suggests that the four rice isoforms are CS genes (Fig. 1). This is confirmed by the presence of a Lys residue, which binds the pyridoxal 5∞-phosphate cofactor (Saito et al., 1993b) and the neighboring residues (PXXSVKDR), which are well conserved among plant CSs (Fig. 1). A molecular phylogenic tree based on the amino-acid sequences of the CSs shows that rcs1 and rcs3 belong to the cytosolic isoform family. Although rcs2 and rcs4 are related to the mitochondrial isoform (cysC family), they are likely to form a new family within the CS superfamily (Fig. 2). A genomic DNA blot analysis was carried out to determine the number of sequences of each isoform. Genomic DNA was digested with BamHI and EcoRI, neither of which has any cleavage sites within the cDNAs used as probes. Genomic DNA was also digested with HindIII, by which rcs1 and rcs3 cDNAs are cut at one site, whereas rcs2 and rcs4 cDNAs are not. The resulting DNAs were then subjected to hybridization analysis with the corresponding cDNA probes (Fig. 3). In addition to single band patterns, additional signals were observed in the BamHI digests of rcs2, in the EcoRI digests of rcs1 and rcs3, and in the HindIII digests of rcs1 and rcs2. This may be due to the presence of cleavage sites within the intron sequences of the genes.
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Fig. 1. Amino-acid alignment of rice CSs. The sequences are rcs1, rcs2, rcs3, rcs4, cytosolic type of Spinacea oleracea (spinach cyt) (Saito et al., 1992), plastidic type (spinach chl ) (Saito et al., 1993a) and mitochondrial type (spinach mit) (Saito et al., 1994). Identical amino-acid residues are shaded. Dashes indicate gaps introduced to optimize the alignment. The asterisks show the conserved region surrounding the pyridoxal 5∞-phosphatebinding Lys residue, which is indicated by the arrow. The sequence data have been deposited in the GenBank, EMBL and DDBJ databases under the Accession Nos AF073695 (rcs1), AF073696 (rcs2), AF073697 (rcs3) and AF073698 (rcs4).
Organ-specific accumulation of transcripts for each isoform in mature plants was examined by RNA blot analysis (Fig. 4). Under the experimental conditions used, cross-hybridization among the four isoforms did not occur (data not shown). The relative levels of the transcripts were estimated by densitometrically quantifying the hybridization intensities, except for rcs4 with an absolutely low transcript level for quantification (Fig. 4B). Transcripts for rcs1 were observed in both shoots and roots, whereas those for rcs2 accumulated only in shoots, and those for rcs3 were found more abundantly in roots than in shoots. The level of rcs4 transcript was very low in all organs tested. The effects of environmental conditions on transcript
accumulation were examined for each isoform (Fig. 5). Seedlings were grown in the 0.5× diluted medium described by Mae and Ohira (1981) for 2 weeks and then grown for 6 days under conditions of sulfur starvation, nitrogen starvation, sulfur and nitrogen starvation, or darkness. Total RNA was isolated from shoots and roots, and the transcript levels of rcs1, rcs2, rcs3 and rcs4 were examined by RNA blot hybridization. The relative levels of the transcripts were estimated by densitometrically quantifying the hybridization intensities, except for rcs4 with an absolutely low transcript level for quantification ( Fig. 5B). Upon sulfur deprivation, the level of rcs1 transcripts was increased in both shoots and roots. However, this response had an absolute
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Fig. 3. DNA blot analysis of rice genomic DNA. Total DNA from rice plants was digested with BamHI ( lane 1), EcoRI ( lane 2) or HindIII ( lane 3). After electrophoretic fractionation and blotting to a nylon membrane, DNA fragments were probed with the corresponding 32P-labeled cDNA. Fig. 2. Phylogenic tree of CSs. The tree was constructed by the neighbor-joining method using CLUSTALW version 1.7 (Thompson et al., 1994) and drawn with TV version 1.5 (Page, 1996). The amino acid sequences are from the rice isoforms (rcs1, rcs2, rcs3, rcs4), Arabidopsis thaliana cytosolic [cytACS1 (Hesse and Altmann, 1995), A.t.OAS.5-8 (Hell et al., 1994)], plastidic [A.t.OAS.7-4 (Hell et al., 1994)], putative mitochondrial (mtACS1, database Accession No. X81973), putative organellar type [CS26 (Nakamura et al., 1997)], Spinacia oleracea (spinach) cytosolic [cysA (Saito et al., 1992)], plastidic [cysB (Saito et al., 1993a)], mitochondrial [cysC (Saito et al., 1994)], Citrullus lanatus (watermelon) cytosolic [cysA (Noji et al., 1994)], Capsicum annuum (bell pepper) chromoplastic [CS C.ann. (Romer et al., 1992)], Triticum aestivum (wheat) cytosolic [cys1 ( Youssefian et al., 1993)], Zea mays (maize) putative cytosolic [Mcysp (Brander et al., 1995)], Solanum tuberosum (potato) cytosolic [StCS-A (Hesse and Hoefgen, 1998)], plastidic [StCS-B (Hesse and Hoefgen, 1998)], Escherichia coli [cysK (Sirko et al., 1990)], and Synechocystis sp. [cysK ( Kaneko et al., 1996)]. The families corresponding to the cysA family, cysB family, cysC family, cs26 family, rcs2 family and bacterial family are indicated by brackets. Numbers along the internodes show the bootstrap probability as a percentage, based on 1000 replicate analyses. The scale indicates the substitutions per site.
requirement for nitrogen, as is clearly shown by the insensitivity to sulfur deprivation in the absence of nitrogen ( Fig. 5B). Also, the response of rcs1 was more sensitive to sulfur deprivation in the roots than in the shoots. Transcript accumulation of rcs3 was generally dependent on light and nutritional deprivation in both shoots and roots. Transcript accumulation of rcs2 was similar to rcs3, except that 6 days of darkness almost completely erased its expression in the shoots. The 109th version of the GenBank database revealed 31 rice EST sequences, of which putative translation products have a high similarity to CSs from plants and bacteria. Twenty-two of these ESTs correspond to the four rice sequences reported here. Nine additional ESTs segregate in four contigs, designated as rcs5, rcs6, rcs7
and rcs8. Each contig was analyzed using the BLASTX program, and the proteins that gave the highest similarity are identified ( Table 1). The Accession Nos for each EST are as follows: rcs1 (D47623, D41715, D47342, D22475, C72939, D41186, D41333, D41092, D41319, D22447, D22394, C97464), rcs2 (D46578, D42350), rcs3 (D47659, C71750, C99640, AU032674, C98455, C99702), rcs4 (D24907, AU031942), rcs5 (C74831, D10957, D48020, D21271), rcs6 (D24896), rcs7 (D23867), rcs8 (C26145, D47368, D47728).
4. Discussion Cysteine synthases of higher plants have been found to constitute several isoforms, which localize to different subcellular compartments. To our knowledge, however, no report has so far been presented on CSs from rice. To investigate the physiological role of rice CS isoforms, we have isolated four cDNAs, rcs1, rcs2, rcs3 and rcs4, that encode CSs. Three of these, rcs1, rcs3 and rcs4, lack an extended N-terminal amino acid sequence ( Fig. 1). This would indicate that rcs1, rcs3 and rcs4 localize to the cytosol, although rcs4, together with rcs2, forms a new CS family. The rcs2 has a putative transit peptide sequence at the N-terminus, but it does not show any obvious features of transit peptides for any organelle. The phylogenic tree ( Fig. 2) indicates that the CSs identified so far are divided into six families, which consist of the cysA family (cytosolic isoform), cysB family (plastidic isoform), cysC family (mitochondrial isoform), cs26 family, rcs2 family, and bacterial family.
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Fig. 4. Organ specificity of transcript accumulation. (A) Total RNA isolated from roots, upper leaves, middle leaves, lower leaves, upper culm, lower culm and panicles of mature rice was fractionated by agarose gel electrophoresis, blotted on to a nylon membrane, and hybridized with 32P-labeled probes. L, lower parts; M, middle parts; U, upper parts. (B) The relative levels of the transcripts were estimated by densitometrically quantifying the hybridization intensities in comparison with those from panicles (100%), except for rcs4 with an absolutely low transcript level for quantification. The intensity of each band was normalized with those of actin bands.
The rcs1 transcript levels were increased within 1 day of sulfur starvation, reaching 1.5- to twofold higher levels after 6 days in comparison with the controls. This response was more pronounced in roots than in shoots. Elevated transcript accumulation in response to sulfur starvation was also observed in other genes involved in the sulfur assimilation pathway, e.g. genes encoding the sulfate transporter (Smith et al., 1995, 1997; Takahashi et al., 1997), ATP sulfurylase (Logan et al., 1996) and APS reductase (Gutierrez-Marcos et al., 1996). This suggests that effective synthesis of cysteine under sulfurdeficient conditions requires the transcriptional
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Fig. 5. Response of transcript accumulation to sulfur, nitrogen and light. Total RNA was isolated from shoots (S ) and roots (R) of plants grown under conditions with full nutrient (C ), sulfur starvation (−S ), nitrogen starvation (−N ), sulfur and nitrogen starvation (−SN ) and darkness (D). Rice seedlings were grown in the 0.5× diluted medium described by Mae and Ohira (1981) at 30°C under continuous light for 2 weeks and subsequently transferred to the indicated condition for 1 week. (A) Total RNA (10 mg) was slot-blotted onto a nylon membrane (Hybond N, Amersham) with a slot-blot apparatus (Millipore), and then hybridized with 32P-labeled probes. (B) Relative mRNA abundance was determined from hybridization intensities of the bands using the BAS-2000 image analyzer (Fuji). The background value, which was calculated from the area just below each bands, was subtracted from the band intensity. Relative mRNA levels are shown in comparison with those from the control conditions of shoots (100%).
induction of a particular CS isoform together with other genes involved in the cysteine synthetic pathway. The induction of rcs1 transcripts by sulfur starvation requires nitrogen to be present in the culture medium.
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Table 1 BLASTX analysis results for contigs from rice ESTs corresponding to cysteine synthase genes Contiga
Number of ESTsb
Similar toc
rcs5 rcs6 rcs7 rcs8
4 1 1 3
Cysteine Cysteine Cysteine Cysteine
synthase synthase synthase synthase
chloroplast precursor chloroplast precursor mitochondrial precursor mitochondrial precursor
Protein IDd
Identity (%)e
Overlapf
P32260 P31300 A55450 A55450
81 82 83 48
215 34 113 89
a Name of the contig (one for each different sequence). b Number of ESTs used to construct each contig. c For which the best similarity was found. d PIR or SwissProt ID of the protein e Percentage of identical residues in the alignment. f Number of amino-acid residues overlapping between a query sequence and its matched protain sequence.
This implies that, in addition to sulfur, rcs1 gene expression is coordinately controlled by nitrogen nutrition, which is necessary to provide one of the substrates for CS, O-acetylserine. Conceivably, rcs1 is involved in the regulatory system that controls the general synthetic balance between sulfur-containing amino acids and other amino acids. The rcs2 transcripts accumulated in green tissues under light, but disappeared completely under dark conditions, indicating that expression of rcs2 is lightdependent. In spinach, synthesis of plastidic CS was induced by light ( Kitamura et al., 1996). Our results together, with these observations, suggest that transcript levels of the rcs2 type CS are concomitantly regulated by photosynthesis, which provides the ATP and reduced ferredoxin necessary for the activation and reduction of sulfate. In addition to an energy supply, photosynthesis also provides carbohydrates with which the carbon skeletons of all amino acids are constructed. The rcs3 transcripts are more abundant in roots than shoots and are reduced under dark conditions, indicating that rcs3 is correlated with photosynthesis in a way similar to that of rcs2. As rcs3 transcript levels were clearly reduced by sulfur and nitrogen deprivation, nutrition and energy possibly regulate its expression in general. The transcript level of rcs4 was very low under all conditions in all organs examined. The expression of rcs4, if any, may be limited to specific cell types and/or environmental conditions. Recently, it has been reported for cocklebur seeds that all cytosolic b-cyanoalanine synthase activity is due to the three cytosolic isoforms of CS (Maruyama et al., 1998). b-cyanoalanine synthase catalyzes the synthesis of b-cyanoalanine from cysteine and cyanide. This implies that multiple CS isoforms are necessary in order to coordinate cysteine and b-cyanoalanine metabolism, although the nature of b-cyanoalanine metabolism is currently unclear. Analyzing rice EST sequences, we identified nine more sequences that showed a high degree of similarity to plant CS ( Table 1). They were grouped into four
contigs, which were designated as rcs5, rcs6, rcs7 and rcs8. The deduced amino-acid sequences from contigs rcs5 and rcs6 revealed a high similarity to plastidic CS, and those from rcs7 and rcs8 had a greater similarity to mitochondrial CS. Contigs rcs5 and rcs7 encode the N-terminal regions, and rcs6 and rcs8 the C-terminal regions of CS, leaving the possibility that they overlap each other. These results indicate that rice contains two to four extra CS genes, in addition to our four genes. However, the exact number of CS isoforms present in any plant is currently not clear. In conclusion, the expression of genes involved in the sulfur assimilation pathway may also depend on the presence of nutrients other than sulfur, nutrient ratios, as well as on light. Further experiments are necessary to understand sulfur metabolism by investigating the spatial gene expression of all CS isoforms under various nutritional conditions.
Acknowledgements The authors thank Drs R. Winz (Max Planck Institute) and T. Kusano (Nara Institute of Science and Technology) for critical reading of the manuscript and suggestion, and the MAFF DNA Bank of National Institute of Agrobiological Resources at Ministry of Agriculture, Forestry and Fisheries of Japan, for the EST clones, D47342, D46578, D47659 and D24907. This work was partly supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture, Japan (06271266) and a grant for Research for the Future Program (JSPS-RFTF 1997R16001) from the Japan Society for the Promotion of Science.
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