Genomic structure and expression analyses of serine acetyltransferase gene in Citrullus vulgaris (watermelon)

Gene 189 (1997) 57–63

Genomic structure and expression analyses of serine acetyltransferase gene in Citrullus vulgaris (watermelon) Kazuki Saito *, Kenji Inoue, Rumiko Fukushima, Masaaki Noji Faculty of Pharmaceutical Sciences, Laboratory of Molecular Biology and Biotechnology in Research Center of Medicinal Resources, Chiba University, Inage-ku, Yayoi-cho 1-33, Chiba 263, Japan Received 1 July 1996; received in revised form 28 October 1996; accepted 29 October 1996

Abstract The genomic clones of Sat gene encoding serine acetyltransferase (SATase), a key enzyme in cysteine biosynthesis in plants, were isolated from the genomic library of Citrullus vulgaris (watermelon). The determination of nucleotide sequence of 5.7 kilobase pair (kbp) length revealed the presence of two introns of 1939 basepair (bp) and 515 bp length in the gene. The transcription start point was determined by primer extension experiments. Southern blot analysis indicated the presence of a single copy of the Sat gene and a couple of additional related sequences in the genome of C. vulgaris. The expression of Sat was analyzed in watermelon plants grown under sulfur- and/or nitrogen-starved conditions and in the presence of pyrazole, O-acetylserine and N-acetylserine. Only slight increment (ca. 1.5–2-fold) of Sat gene expression was observed upon sulfur starvation for 48 h. Interestingly, the addition of pyrazole, which is a precursor of b-pyrazolealanine (b-PA) synthesized by SATase and cysteine/b-PA synthase, enhanced the expression of Sat by ca. 2-fold. © 1997 Elsevier Science B.V. Keywords: Cysteine biosynthesis; Sulfur nutrition; b-Pyrazolealanine; Amino acid metabolism

1. Introduction Serine acetyltransferase (SATase) (EC 2.3.1.30) plays a key role in the biosynthesis of cysteine in plants and in bacteria. This enzyme catalyzes the formation of Oacetylserine (OAS ) from serine and acetyl-CoA, and thus it is placed at the conjunction step between serine metabolism and cysteine biosynthesis (Saito, 1995). It has been indicated that the supply of OAS is a ratelimiting factor in cysteine biosynthesis in vivo (Saito et al., 1994). OAS is the precursor of b-pyrazolealanine (b-PA), a secondary product found in Curcurbitaceae plants, as well as the precursor of cysteine (Brown, 1993). Recently, the cDNAs encoding SATase have been * Corresponding author. Tel. +81 43 2902904; Fax +81 43 2902905; e-mail: [email protected] Abbreviations: bp, base pair(s); cDNA, DNA complementary to RNA; CSase, cysteine/b-PA synthase (O-acetylserine(thiol )-lyase); Cys, gene encoding CSase; kb, kilobase(s) or 1000 bp; NAS, N-acetylserine; OAS, O-acetylserine; Sat, gene encoding SATase; SATase, serine acetyltransferase; tsp, transcription start point(s); UTR, untranslated region(s); b-PA, b-pyrazolealanine. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 8 33 - 5

isolated from Citrullus vulgaris (watermelon) (Saito et al., 1995) and Arabidopsis thaliana (Bogdanova et al., 1995; Hell and Bogdanova, 1995; Murillo et al., 1995; Ruffet et al., 1995; Roberts and Wray, 1996). The recombinant SATase of watermelon produced in Escherichia coli formed a multienzyme complex with cysteine/b-PA synthase [O-acetylserine(thiol )-lyase] (CSase) ( EC 4.2.99.8) (Saito et al., 1995). CSase catalyzes the formation of cysteine and b-PA, the terminal step of cysteine/b-PA biosynthesis. The activity of the purified recombinant SATase of watermelon was subjected to a feedback inhibition by cysteine (Saito et al., 1995), an end-product of the biosynthetic pathway, but not by b-PA, the other final product of the biosynthetic pathway in watermelon. These previous investigations suggested that SATase plays a regulatory role at the level of enzyme activity in cysteine biosynthesis. However, the involvement of transcriptional control of the Sat gene encoding SATase still remains to be clarified. So far, no information has been available about the genomic structure of the Sat gene and the expression pattern under some nutritional stress in plants. In the present study, we have determined the structure of the Sat gene in watermelon and elucidated the

58

K. Saito et al. / Gene 189 (1997) 57–63

accumulation pattern of the Sat mRNA under some nutritional conditions and in the presence of additives.

2. Experimental and discussion

gent washing conditions ( Fig. 2). The single copy band was detected under the high stringency. However, the presence of 2 or 3 bands was shown by the low-stringent washing. These indicated the presence of a single copy gene encoding the SATase and also the presence of 2 or 3 copies of a sequence homologous to the Sat gene.

2.1. Isolation and structure of the Sat gene 2.2. Structural feature of promoter region of the Sat gene Screening of lEMBL3 genomic library (2.7×105 independent clones) prepared from genomic DNA of C. vulgaris by the cDNA encoding SATase (Saito et al., 1995) allowed isolation of seven positive phage clones. By the restriction analysis and cross-hybridization experiments for seven isolated clones, a clone, gSAT2, whose insert covered the entire region of the Sat gene, was selected for sequence determination. The sequence analysis of the region spanning 5.7 kb in the insert of gSAT2 revealed that the Sat gene comprises three exons and two introns of 1939 bp and 515 bp length, respectively, deduced by comparing with the cDNA sequence (Fig. 1). The all exon-intron junctions are conforming to ‘GT-AG rule’ for splicing donor and acceptor sites (Lewin, 1990). Intron 1 is located just 3 bp 5∞ upstream of the first ATG for translational start codon in the cDNA. The sequence of exon 2 encodes the less conserved N-terminal region of the SATase protein among several SATase proteins reported; whereas the translational domain encoded by exon 3 contains the motif postulated as the binding site of acetyl-CoA, which is highly conserved among several different types of acetyltransferase not only for SATases. These suggested some molecular evolutionary implication of genes and proteins for acetyltransferase group by exon shuffling (Gilbert, 1978). The sequence determination clarified the presence of the in-frame stop codon in the 5∞-untranslated region (UTR) with respect to the initial ATG deduced in the cDNA sequence (Saito et al., 1995), confirming the identity of this ATG as the translational start codon. Southern blot analysis on the genomic DNA of watermelon was carried out under high- and low-strin-

Transcriptional start point (tsp) was determined by primer extension experiments as shown in Fig. 3. The tsp is at A1 located at 416 bp 5∞-upstream of intron 1 ( Fig. 1). Two TATA box and CAAT box-like sequences (underlining) were found at −22 bp, −36 bp and −164 bp positions, respectively, from the tsp. There are two sets of direct repeat (arrowed underlining) of 10 bp and 11 bp length, respectively, and an inverted repeat (dotted underlining) of 11 bp length in the promoter region within −1 kbp. Two palindrome sequences (double underlining) of 14 bp and 10 bp length are also found in this 5∞-upstream region. It is of interest that the two probable cis-element-like structures which are identical or very similar to SEF 4 recognition sequence were found in this region (asterisked). SEF 4 is a transfactor which is believed to respond to sulfur deficiency and to be involved in the gene expression for Met-rich storage protein, b-conglycinin, in soybean (Allen et al., 1989; Lessard et al., 1991). 2.3. Expression pattern of Sat and Cys under sulfur- and nitrogen-starved conditions Watermelon seedlings were grown in the control medium for 14 days, and then the medium was changed to those of sulfate deficiency (S−), nitrogen deficiency (N−), and sulfate and nitrogen double deficiency (S−, N−). The change of mRNA levels of Sat and Cys encoding CSase were determined by Northern blot analysis for the total RNA prepared from the plants harvested by each 24 h interval (Fig. 4). The mRNA accumulations of Sat in the control

Fig. 1. Organization of the Sat gene in C. vulgaris (watermelon). (A) Schematic representation of the structure and partial restriction map of the Sat gene and the cDNA. Filled box represents protein coding region in exons. Hatched box represents 5∞- and 3∞-UTRs. Sc, SacI; E, EcoRI; H, HindIII; S, SalI. (B) Nucleotide and deduced amino acid sequence of the Sat gene in gSAT2 clone. The nucleotides in the cDNA are expressed in upper case. The nucleotides in 5∞- and 3∞-flanking regions and introns are shown in lower case. Tsp determined by the experiments in Fig. 4 is indicated at the position 1. The first nucleotide position, C377, in the cDNA clone, pSAT2 (Saito et al., 1995), is indicated by a dot. Underlining indicates TATA box and CAAT box like sequences. Arrowed underlining indicates direct repeat. Dotted and arrowed underlining indicates inverted repeat. Double underlining indicates palindrome structure. Asterisk indicates SEF 4 recognition like sequence (A/GTTTTTA/G). The position of R4 primer used for the primer extension assay is indicated by dotted underlining. The entire sequence reported here has been deposited in the DDBJ/EMBL/GenBank data base as accession No. D85624. Methods: For the construction of genomic library, the genomic DNA isolated from C. vulgaris cv. Kinro (Sataka, Yokohama, Japan) was partially digested with Sau3AI, treated with alkaline phosphatase, and then ligated into BamHI site of lEMBL3 vector (Stratagene, La Jolla, CA, USA). The constructed phage library with E. coli XL1-Blue MRA (P2) (2.7×105 independent plaques) was screened with 32P-labeled SATase cDNA from C. vulgaris carried on pSAT2 as the probe. Among 7 isolated clones, gSAT was selected by the restriction analysis and cross hybridization experiments using 5∞- and 3∞-terminal regions as probes for further analysis. The insert in gSAT2 was digested with restriction enzymes and sub-cloned into several overlapping plasmids on pBluescript II SK−. The sequence of the insert in the plasmid vectors were determined for both strands with a series of deletion clones.

K. Saito et al. / Gene 189 (1997) 57–63

59

60

K. Saito et al. / Gene 189 (1997) 57–63

Fig. 2. Southern blot analysis of genomic DNA of watermelon. Total DNA (20 mg each) isolated from C. vulgaris cv. Kinro seedlings was digested with the restriction enzymes, separated by agarose gel (0.8%) electrophoresis and transferred onto a nylon membrane (Hybond N+, Amersham, Amersham, UK ). The DNA blotted on the filter was hybridized with the random-prime-labeled Sat cDNA carried on pSAT2 as the 32P-probe as reported previously (Saito et al., 1995). (A) After low stringent washing: The final wash of the filter was performed in 2×SSPE and 0.1% SDS at 65°C for 15 min. (B) After high stringent washing: The filter was washed further in 0.1×SSPE and 0.1% SDS at 65°C for 15 min.

Fig. 3. Determination of tsp by primer extension analysis. Primer extension assay was carried out essentially as described previously (McKnight and Kingsbury, 1982). Briefly, total RNA (40 mg) prepared from etiolated seedlings was annealed with R4 primer (30-mer, 10 pmol ) whose sequence is indicated in Fig. 4. Reverse transcription was performed by M-MLV reverse transcriptase (60 units) in the presence of [32P]dCTP (370 kBq) and dNTPs (2 mM each) in 50 ml. The primer extension product ( lane 5) was separated on a polyacrylamide (5%) sequencing gel together the sequencing products ( lanes 1–4) of M13mp18 DNA as the template with M13 P4 oligonucleotide (17-mer) as the primer for the size marker.

medium decreased slightly, probably because of the ‘transfer effect’ into the new medium. Under the sulfurstarved condition, the accumulation of Sat mRNA increased in comparison with that in the control medium; evidently, the mRNA level after 48 h starvation was enhanced to ca. 1.5–2-fold of that in the control

medium at the same time. Recently, it has been shown coincidentally that the SATase mRNA level in A. thaliana increased 1.5–2-fold by sulfur depletion stress (Bogdanova et al., 1995). In contrast, nitrogen starvation even weakly decreased the Sat mRNA accumulation. Double starvation of sulfur and nitrogen did not change the level of Sat transcript. The mRNA level of Cys was not affected by sulfur starvation and even down-regulated by nitrogen depletion and double starvation of sulfur and nitrogen. These results indicated that the steady-state level of Sat mRNA is up-regulated by nutritional sulfur supply by a factor of 1.5–2; however, the Cys mRNA level is less controlled by sulfur nutrition. In Spinacia oleracea (spinach) cell cultures, sulfur depletion induced very weakly (1.2–1.5-fold) the mRNA levels of CysA encoding the cytoplasmic CSase and CysB encoding the chloroplastic isoform ( Takahashi and Saito, 1996). Since the Cys gene studied in the present investigation also encodes the cytoplasmic isoform of CSase in watermelon (Saito et al., 1995), the weak response towards sulfur starvation seems to be a common feature of Cys gene expression in plant cells. 2.4. Induction of the Sat mRNA accumulation by addition of pyrazole, the precursor of b-PA To clarify the effect of additives on the expression of Sat gene, the Northern blot analysis was carried out with the RNA from the seedlings grown in the media supplemented with pyrazole, OAS and N-acetylserine (NAS) ( Figs. 5 and 6). Pyrazole is the biosynthetic precursor of b-PA, which is produced from pyrazole and OAS by the action of CSase in watermelon (Noji et al., 1993, 1994). Pyrazole exhibited the induction effect on the Sat gene expression by a dose-dependent manner. At the concentration of 20 mM pyrazole, the mRNA level of Sat after 48 h induction was 2-fold higher than that of control of no induction. In contrast, the induction effect of pyrazole on the Cys expression was less than that on the Sat expression. The increased mRNA accumulation of Sat could result in the higher production of OAS needed for conjugation reaction with pyrazole. It was reported that CSase is present in higher concentration than SATase in the cells by a specific activity basis (Ruffet et al., 1994), suggesting a sufficient amount of CSase for b-PA formation in an overloaded concentration of pyrazole. Thus, the enhanced accumulation of Sat transcript is presumably required for the formation of b-PA from supplemented pyrazole. It should be noted that the feeding of 20 mM pyrazole to Cucumis sativus seedlings resulted in an approximately 2-fold increment of b-PA production (Brown and Diffin, 1990), concomitant with the induction effect on the Sat gene expression. OAS is the product of the reaction catalyzed by

K. Saito et al. / Gene 189 (1997) 57–63

61

Fig. 4. Change of mRNA accumulation of Sat and Cys under sulfur and/or nitrogen starvation. The time-course of mRNA accumulation of Sat (A) and Cys (B) after transfer to control (%), S− (n), N− ($) and S−, N− (+) media. The watermelon seedlings were grown with the control medium (1.5 mM MgSO , 2 mM Ca(NO ) , 3 mM KNO 1.5 mM NaH PO , 0.26 mM Na HPO , 8.7 mM NaFeEDTA, 10.3 mM MnCl , 1.0 mM 4 32 3, 2 4 2 4 2 ZnCl , 0.96 mM CuCl , 30 mM H BO , 24 nM (NH ) Mo O , 130 mM CoCl ) for 14 days at 22–25°C under daylight, and then the medium was 2 2 3 3 46 7 24 2 changed to the fresh control medium or to the nutrient-deficient media; sulfate-deficient (S−) medium by replacing MgSO with MgCl , nitrate4 2 deficient (N−) medium by replacing the nitrate ions with the corresponding chloride ions, and both sulfate-deficient and nitrate-deficient (S−, N−) medium. Total mRNA was extracted by every 24 h interval after changing the medium. The isolated RNA was separated by denatured agarose gel electrophoresis, transferred onto nylon membranes, and then hybridized with 32P-labeled cDNA probes for Sat (Saito et al., 1995) and Cys (Noji et al., 1994) on the filter, respectively, as described elsewhere (Saito et al., 1995). Hybridization with rice rDNA as the internal load control was also carried out to normalize the amounts of RNA blotted on the filter. The final wash of the filter was performed in 0.1×SSPE and 0.1% SDS at 65°C for 15 min. Hybridization signals were quantified by BAS-2000 image analyzer (Fuji Film) and normalized towards the signals of ribosomal RNA as the load control.

effects, at least, on the accumulation of Sat and Cys mRNAs in watermelon. These results indicated that the regulation of Sat gene expression is mediated by the concentration of pyrazole as well as the supply of nutritional sulfur. Presumably, some cis elements located in the 5∞-upstream region are responsible for the regulation of such gene expression. With respect to the cis elements responsible for sulfur starvation, the sequences similar to SEF 4 recognition sequence identified within the −1 kb region would be of interest for further analysis of the molecular dissection of the promoter region. Fig. 5. Representative Northern analysis for the change of mRNA accumulation of Sat and Cys by addition of pyrazole. The watermelon seedlings were grown with the control medium for 14 days at 22–25°C under daylight, and then the medium was changed to the new media with no supplement control and supplemented with 0.5 mM, 5 mM or 20 mM pyrazole. Northern blot analysis were carried out using the 32P-labeled cDNAs for Sat, Cys and rDNA as probes as described in the legend to Fig. 4.

SATase, and NAS is an isomer of OAS formed by rearrangement of acetyl group from the oxygen atom to the nitrogen atom. OAS and NAS are proven to be the inducers of gene expression involved in cysteine biosynthesis in Salmonella typhimurium (Ostrowski and Kredich, 1989). However, OAS and NAS exhibited no

3. Conclusions (1) We have determined the genomic structure of the Sat gene encoding SATase in C. vulgaris (watermelon). The Sat gene, extending ca. 5 kb in length, comprises three exons and two introns. A single copy of Sat gene is present in the genome of C. vulgaris. (2) The expression of Sat gene is up-regulated by sulfur starvation by a factor of 1.5–2. However, the level of Cys transcript encoding CSase is not substantially subject to sulfur nutritional control. (3) Pyrazole, the precursor of b-PA, induces the Sat

62

K. Saito et al. / Gene 189 (1997) 57–63

Fig. 6. Change of mRNA accumulation of Sat and Cys by addition of pyrazole, OAS or NAS. The time-course of mRNA accumulation of Sat (A and C ) and Cys (B and D) after transfer to the media supplemented with pyrazole (A and B), OAS or NAS (C and D). The watermelon seedlings were grown with the control medium for 14 days at 22–25°C under daylight, and then the medium was changed to the new media with no supplement control (%), supplemented with 0.5 mM (n), 5 mM ($) or 20 mM (+) pyrazole, or 0.5 mM OAS ()) or 0.5 mM NAS (#). Northern blot analysis and quantification of the signals were carried out as described in the legend to Fig. 4.

gene expression by ca. 2-fold, presumably responding to the demand of OAS supply necessary for the formation of b-PA from pyrazole.

Acknowledgement This research was supported, in part, by Grants-inAids from the Ministry of Education, Science, Sports, and Culture, Japan, and by the Research for the Future Program (96I00302) from the Japan Society for the Promotion of Science.

References Allen, R.D., Bernier, F., Lessard, P.A. and Beachy, R.N. (1989) Nuclear factors interact with a soybean b-conglycinin enhancer. Plant Cell 1, 623–631. Bogdanova, N., Bork, C. and Hell, R. (1995) Cysteine biosynthesis in plants: isolation and functional identification of a cDNA encoding a serine acetyltransferase from Arabidopsis thaliana. FEBS Lett. 358, 43–47. Brown, E.C. and Diffin, F.M. (1990) Biosynthesis and metabolism of

pyrazole by Cucumis sativus: enzymic cyclization and dehydrogenation of 1,3-diaminopropane. Phytochemistry 29, 469–478. Brown, E.G. (1993) Enzymes of N-Heterocyclic Ring Synthesis. In: Lea, P.J. ( Ed.), Methods in Plant Biochemistry 9, Enzymes of Secondary Metabolism. Academic Press, London, pp. 153–181. Gilbert, W. (1978) Why genes in pieces? Nature 271, 501. Hell, R. and Bogdanova, N. (1995) Characterization of a full-length cDNA (Accession No. X80938) encoding serine acetyltransferase from Arabidopsis thaliana. (PGR95-108). Plant Physiol. 109, 1498. Lessard, P.A., Allen, R.D., Bernier, F., Crispino, J.D., Fujiwara, T. and Beacy, R.N. (1991) Multiple nuclear factors interact with upstream sequences of differentially regulated b-conglycinin genes. Plant Mol. Biol. 16, 397–413. Lewin, B. (1990) Gene IV. Oxford University Press, Oxford. McKnight, S.L. and Kingsbury, R. (1982) Transcriptional control signals of a eukaryotic protein-coding gene. Science 217, 316–324. Murillo, M., Foglia, R., Diller, A., Lee, S. and Leustek, T. (1995) Serine acetyltransferase from Arabidopsis thaliana can functionally complement the cysteine requirement of a cysE mutant strain of Escherichia coil. Cell. Mol. Biol. Res. 41, 425–433. Noji, M., Murakoshi, I. and Saito, K. (1993) Evidence for identity of b-pyrazolealanine synthase with cysteine synthase in watermelon: formation of b-pyrazolealanine by cloned cysteine synthase in vitro and in vivo. Biochem. Biophys. Res. Commun. 197, 1111–1117. Noji, M., Murakoshi, I. and Saito, K. (1994) Molecular cloning of a cysteine synthase cDNA from Citrullus vulgaris (watermelon) by genetic complementation in an Escherichia coli Cys− auxotroph. Mol. Gen. Genet. 244, 57–66.

K. Saito et al. / Gene 189 (1997) 57–63 Ostrowski, J. and Kredich, N.M. (1989) Molecular characterization of the cysJIH promoters of Salmonella typhimurium and Escherichia coli: regulation by cysB protein and N-acetyl--serine. J. Bateriol. 171, 130–140. Roberts, M. A. and Wray, J. L. (1996) Cloning and characterization of an Arabidopsis cDNA clone encoding an organellar isoform of serine acetyltransferase. Plant Mol. Biol. 30, 1041–1049. Ruffet, M.-L., Droux, M. and Douce, R. (1994) Purification and kinetic properties of serine acetyltransferase free O-acetylserine(thiol )lyase from spinach chloroplasts. Plant Physiol. 104, 597–604. Ruffet, M.-L., Lebrun, M., Droux, M. and Douce, R. (1995) Subcellular distribution of serine acetyltransferase from Pisum sativum and characterization of an Arabidopsis thaliana putative cytosoli isoform. Eur. J. Biochem. 227, 500–509. Saito, K. (1995) Cysteine biosynthesis as a sulfur assimilation pathway

63

in plants: molecular and biochemical approach. In: Mathis, P. (Ed.), Photosynthesis: From Light to Biosphere. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 347–352. Saito, K., Kurosawa, M., Tatsuguchi, K., Takagi, Y. and Murakoshi, I. (1994) Modulation of cysteine biosynthesis in chloroplasts of transgenic tobacco overexpressing cysteine synthase [O-acetylserine(thiol )-lyase]. Plant Physiol. 106, 887–895. Saito, K., Yokoyama, H., Noji, M. and Murakoshi, I. (1995) Molecular cloning and characterization of a plant serine acetyltransferase playing a regulatory roles in cysteine biosynthesis from watermelon. J. Biol. Chem. 270, 16321–16326. Takahashi, H. and Saito, K. (1996) Subcellular localization of spinach cysteine synthase isoforms and regulation of their gene expression by nitrogen and sulfur. Plant Physiol. 112, 273–280.