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Sulfur assimilation in soybean: molecular cloning and characterization of O-acetylserine (thiol) lyase (cysteine synthase).

SOYBEAN TYPICALLY CONTAINS about 40% protein and 20% oil. Because of their high protein content, they are widely used as a protein source both for humans and animals. In the USA, soybeans are mainly used as animal feed. Soybean proteins are deficient in sulfur-containing amino acids (methionine and cysteine). Unlike plants, animals have a dietary requirement for sulfur amino acids. As a consequence, animal diets are often supplemented with synthetic sulfur amino acids to achieve optimal growth. The use of supplemental amino acids costs the poultry and swine industry approximately 100 million dollars annually. Therefore, improving the sulfur-amino acid content of soybean proteins will greatly benefit the livestock industry and improve the overall profitability of soybean farmers.

Soybean seed proteins are classified into 7S and 11S proteins and these together represent about 70% of the total seed protein (Nielsen, 1996; Krishnan, 2000). The 11S proteins are named glycinin, while the 7S proteins are known as [beta]-conglycinin. The 11S glycinin contains more of the sulfur-containing amino acids than the 7S [beta]-conglycinin. The [beta]-conglycinin is made up of three subunits, namely [alpha]', [alpha], and [beta]. The [beta]-subunit lacks methionine (Coates et al., 1985) and is considered to be of very poor nutritional quality. Elimination or reduction of the [beta]-subunit of [beta]-conglycinin, therefore, may lead to improvement of the nutritional quality of soybean seed proteins. In fact, soybean seed storage protein mutants have been obtained that have low levels of 7S globulins and such mutants have 15% more methionine than other cultivars (Kitamura and Kaizuma, 1981). Recently, Imsande (2001) reported the isolation of soybean mutant lines with a methionine over-producing phenotype. It was reported such mutants had approximately 20% greater methionine and cysteine concentration in their seeds. The nutritional quality of soybean seed storage proteins has also been enhanced by expressing heterologous proteins that are rich in methionine. Introduction of methionine-rich 2S albumin from Brazil nut (Bertholletia excelsa Humb. & Bonpl.) drastically improved the methionine content of soybean (Nordlee et al., 1996). However, the introduced Brazil nut protein was an allergen, and consequently, the transgenic soybeans were not commercialized. Interestingly, transgenic soybeans expressing Brazil nut 2S albumin showed lower accumulation of Kunitz trypsin inhibitor (KTI) and chymotrypsin inhibitor (CI). The protease inhibitors are rich in sulfur-containing amino acids and the heterologous expression of 2S Brazil nut albumin has presumably shunted the sulfur amino acids from the protease inhibitors (Streit et al., 2001). This study indicates that there is a limitation in the sulfur amino acid pool in developing soybean seeds.

We are interested in improving the protein quality of soybean seed storage proteins. One of the approaches that we have undertaken is genetic manipulation of enzymes that are involved in the sulfur biosynthetic pathway. In spite of the importance of sulfur amino acids in determining the protein quality of soybean, we know very little about sulfur metabolism in soybean. Our objective was to identify and characterize enzymes involved in cysteine biosynthetic pathway in soybeans. The cysteine biosynthetic pathway is responsible for the fixation of inorganic sulfur into L-cysteine (Leustek et al., 2000). Cysteine is the first reduced sulfur-containing compound and serves as the sulfur donor for methionine. The cysteine biosynthetic pathway involves several enzymatic steps (Leustek and Saito, 1999). O-acetylserine (thiol) lyase [OAS-TL; EC 4.2.99.8] catalyzes the formation of cysteine from O-acetylserine (OAS) and hydrogen sulfide with the release of acetic acid.

OAS + [H.sub.]2S [right arrow] cysteine + acetic acid

Cysteine is the principal starting material for the synthesis of sulfur-containing amino acids, coenzymes, and sulfolipids (Leustek et al., 2000). In spite of the importance of this enzyme in sulfur metabolism, no molecular characterization of this enzyme in soybean has been reported. Here, we report the molecular cloning and characterization of OAS-TL from soybean, an enzyme that catalyzes the final step of the cysteine biosynthetic pathway.

MATERIALS AND METHODS

Plant Material

Soybean cv. Williams 82 was field-grown at the Bradford Research and Extension Center near Columbia, MO, on a Mexico silt loam soil (Udollic Ochraqualf). Samples were collected from nodes 10 and 11 over time starting from the R5 stage (Fehr and Caviness, 1979). Seeds from four replications were collected for each time point. The pod walls were split open and seeds were frozen immediately in liquid nitrogen and stored at -80[degrees]C until use.

cDNA Isolation and Sequence Analysis

Total RNA from developing soybean seeds was isolated by the LiCl precipitation procedure (Lizzardi, 1983). Poly [(A).sup.+] RNA was isolated by oligo (dT)-cellulose chromatography. A cDNA library of soybean seed poly [(A).sup.+] RNA was constructed in lambda ZAP II following the manufacturer's protocol (Stratagene, La Jolla, CA). To isolate the OAS-TL cDNA clone, we synthesized primers (Forward: 5' GGGTTACAAG CTCATAATTAC 3'; Reverse: 5' GCACCTGTCATTGTA CCACGAG 3') on the basis of published sequence of a EST clone (GenBank accession no. AW509442). These primers were utilized to amplify a 300-bp fragment from soybean genomic DNA. The polymerase chain reaction (PCR) fragment was purified from agarose gel and radiolabeled with [[alpha]-[sup.32]P]dCTP (PerkinElmer Life Sciences Inc., Boston, MA) with a random labeling kit (Takara Bio Inc., Shiga, Japan). The soybean seed cDNA library was screened with the radiolabeled probe according to standard protocol (Sambrook et al., 1989). Three positive lambda clones were identified by colony hybridization and the plasmids from these clones were recovered with the use of the Rapid Excision Kit (Stratagene, La Jolla, CA). One of the positive clones (pSCS1) was chosen for further analysis. The cDNA insert was sequenced with a Taq Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) at the DNA core facility of the University of Missouri using appropriate primers synthesized by Integrated DNA Technologies (Coralville, IA). The nucleotide sequence and the derived amino acid sequence of soybean seed OAS-TL were subjected to BLAST analysis (BLASTX, National Center for Biotechnology Information-NCBI). Restriction enzyme analysis was performed by means of NEBCutter 1.0 (New England Biolabs, Beverly, MA). Multiple sequence alignments were performed by CLUSTLAW software (European Bioinformatics Insitute, Germany; http://www.ebi.ac.uk/clustalw; verified 7 March 2003) and BOXSHADE (Pasteur Institute, France; http://bioweb.pasteur.fr/seqanal/interfaces/boxshade.html; verified 7 March 2003). A phylogenic tree was constructed with the help of the University of California database (University of California, San Diego, CA; http://workbench.sdsc.edu; verified 7 March 2003).

Isolation of Soybean Genomic DNA and Southern Blotting

Genomic DNA from soybean leaf (cv. Williams 82) was isolated by the standard hexadecyltrimethylammonium bromide (CTAB) method. Ten micrograms of genomic DNA were digested with BamHI, EcoRI, and HindIII overnight at 37[degrees]C. The digested samples were fractionated on a 0.8% (w/v) agarose gel. After electrophoresis, the DNA was partially hydrolyzed (15 min depurination in 0.25 M HCl; 30 min denaturation in 0.4 mol [L.sup.-1] NaOH) before transfer to Hybond [N.sup.+] membrane (Amersham Biosciences, Piscataway, NJ). After transfer, the filter was prehybridized overnight in hybridization buffer at 65[degrees]C (100 g [kg.sup.-1] BSA, 500 mmol [L.sup.-1] [Na.sub.2]HP[O.sub.4], 10 mmol [L.sup.-1] EDTA, 70 g [kg.sup.-1] SDS, 100 mg [L.sup.-1] salmon sperm DNA, total volume of 20 mL). Hybridization was performed at 65[degrees]C for 24 h with [[alpha]-[sup.32]P]dCTP labeled OAS-TL probe. Following hybridization, the membrane was washed two times with 2x SSC (1x SSC is 0015 M NaCl plus 0.015 M sodium citrate), 10 g [kg.sup.-1] SDS, once with 1x SSC, 10 g [kg.sup.-1] SDS and finally two more times with 0.1x SSC, 10 g [kg.sup.-1] SDS. Each wash was performed for 10 min at 65[degrees]C. Hybridizing bands were detected by autoradiography, using a DuPont (Wilmington, DE) Cronex Lightening Plus intensifying screen for signal enhancement.

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Analysis

Total RNA from developing soybean seeds was extracted by means of Trizol Reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Total RNA (0.1 [micro]g) was used for the reverse transcriptase (RT) reaction. Before RT-PCR, the RNA was treated with DNase I (Invitrogen, Carlsbad, CA) to remove any contaminating DNA. The RT reaction was performed in a volume of 50 [micro]L with the OneStep RT-PCR kit (Qiagen, Valencia, CA). Primers were designed from the 5' end and 3' end of the open-reading frame (ORF) of OAS-TL. The forward and reverse primers were 5'-CCAAC ATATGATGGCTGTTGAAAGGTCCGG-3' and 5'-GGT TGCGGCCGCTCAGGGCTCAAAAGTCATGC-3', respectively. The thermal cycler program was 50[degrees]C for 30 min, 95[degrees]C for 15 min, 30 cycles at 94[degrees]C (1 min), 58[degrees]C (1 min), and 72[degrees]C (1 min), followed by a final 10 min at 72[degrees]C. A 700-bp fragment of soybean 18S rRNA was also reverse-transcribed under similar conditions and used as a loading control. Primer sequences were as follows: Forward: 5'-GCTTAACACATGCAAGTC GAACGTTG-3', Reverse: 5'-ACCCCTACACACGAAAT TCCACTC-3'. The PCR products were separated on a 7 g [kg.sup.-1] agarose gel and photographed with an Eagle Eye II still video system (Stratagene, La Jolla, CA).

Western Blot Analysis

Seed proteins isolated from soybeans at different developmental stages were separated by SDS-PAGE (Laemmli, 1970) with a Mighty Small II electrophoresis system (Hoefer Scientific Instruments, San Francisco, CA). The proteins were resolved on a slab gel (10 x 8 x 0.75 cm) consisting of a 135 g [kg.sup.-1] separation gel and a 40 g [kg.sup.-1] stacking gel. Electrophoresis was performed at 20 mA constant current per gel at room temperature. After the completion of the electrophoresis, the gels were equilibrated with electrode buffer (25 nmol [L.sup.-1] Tris, 192 mmol [L.sup.-1] glycine, and 200 g [kg.sup.-1] methanol, pH 8.3) for 15 min. Proteins from the gels were electroblotted onto pure nitrocellulose membrane (Midwest-Scientific, Valley Park, MO) essentially as described by Burnett (1981). The membranes were washed with TBS (80 mmol [L.sup.-1] Tris-HCl, 200 mmol [L.sup.-1] NaCl, pH 7.5) for 5 min and incubated with TBS containing 50 g [kg.sup.-1] nonfat dried milk for 1 h at room temperature. Following this, the membrane was incubated overnight with polyclonal antibodies raised against soybean recombinant OAS-TL (Chronis and Krishnan, unpublished) that was diluted 1:5,000 in TBS containing 50 g [kg.sup.-1] nonfat dried milk. Following three washes in TBS containing 0.8 g [L.sup.-1] of Tween 20 (TBST) for 10 min each, the blot was incubated with HRP-conjugated goat anti-rabbit IgE (1:5,000 [v/v] dilution) in TBST containing 50 g [kg.sup.-1] nonfat dried milk for 1 h with gentle agitation at room temperature. Final washes were performed with TBST (3 x 10 min) and TBS (1 x 5 min). Immunoreactive polypeptides were visualized by horseradish peroxidase color development procedure recommended by the manufacturer (Bio-Rad Laboratories, Richmond, CA).

Complementation of NK3 [Cys.sup.-] E. coli Auxotroph

We amplified the coding region of soybean OAS-TL with gene-specific primers (Forward 5'-CCAACATATGATGGC TGTTGAAAGGTCCGG-3' and Reverse 5'-GGTTGCGG CCGCTCAGGGCTCAAAAGTCATGC-3') to which NotI and NdeI restriction sites were introduced at the 5'and 3' ends, respectively, The PCR product was cloned into the NdeI and NotI sites of the expression vector pET 28(a)+ (Calbiochem-Novabiochem, San Diego, CA) resulting in pSCS10. The NK3 [Cys.sup.-] E. coli mutant [[DELTA]trpE5 leu-6 thi hsdR hsd[M.sup.+] cysK cysM], (obtained from Dr. Kazuki Saito, Chiba University, Chiba, Japan) was transformed with pSCS10 and the cloning vector pET-28a served as a negative control. For the genetic complementation of the cysteine requirement, the transformed E. coli ceils were plated on M9 agar plates (Sambrook et al., 1989) supplemented with 100 mg [L.sup.-1] kanamycin, 1 mmol [L.sup.-1] IPTG and 0.2 g [kg.sup.-1] leucine and tryptophan (Saito et al., 1992).

Determination of OAS-TL Activity

The OAS-TL activity was determined according to the protocol of Warrilow and Hawkesford (1998). Soybean seed samples (200 mg) were ground in a chilled mortar and pestle with 2 mL of ice-cold extraction buffer [100 mmol [L.sup.-1] Tris-HCl pH 8.0, 100 mmol [L.sup.-1] KCl, 20 mmol [L.sup.-1] Mg[Cl.sub.2], 10 g [kg.sup.-1] Tween 80and 10mmol [L.sup.-1] dithiothreitol (DTT)]. The samples were transferred to microcentrifuge tubes and centrifuged at 4[degrees]C for 10 min at 12 000 g. The clear supernatant was saved and used immediately for measuring the OAS-TL activity. Protein concentrations from seed extracts were determined spectrophotometrically with the help of DC Standard Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA). The enzyme reaction mixture contained 5 mmol [L.sup.-1] OAS, 3 mmol [L.sup.-1] sodium sulphide, 10 mmol [L.sup.-1] DTT and 0.1 tool [L.sup.-1] sodium phosphate pH 8 in total volume of 0.2 mL. The reaction was initiated by the addition of OAS and the mixture was incubated at 26[degrees]C for 10 min. After the incubation period, 0.15 mL aliquots were removed and mixed with 0.35 mL of acidic ninhydrin reagent (13 g [kg.sup.-1] ninhydrin in 1:4 HCl: HOAc) and heated at 100[degrees]C for 10 min to allow color development. After cooling on ice, 0.7 mL of ethanol were added and absorbance measured at 550 nm. One unit of enzyme activity is defined as the conversion of 1 nmol of OAS into cysteine per minute under the stated assay conditions. Assays were performed three times and each time was represented by two replications.

RESULTS

Isolation of a cDNA Encoding OAS-TL from a Soybean Seed cDNA Library

To isolate the OAS-TL cDNA clone from soybean seed cDNA library, we synthesized primers corresponding to 5' and 3' of an EST clone (AW509442) encoding OAS-TL. These primers were utilized to amplify a 300-bp fragment from soybean genomic DNA. This PCR product was labeled with [sup.32]P and used as a hybridization probe to screen a soybean seed cDNA library constructed in lambda ZAP II vector resulting in the isolation of three putative clones. Subsequent restriction enzyme digestion of the DNA isolated from the three positive cDNA clones showed the same restriction pattern for all of them, and one of these clones (PCS1) was chosen for further studies. The physical map of this cDNA clone is shown in Fig. 1A. To characterize the putative OAS-TL cDNA clone, the nucleotide sequence was determined at the DNA core facility of the University of Missouri. The nucleotide sequence revealed that the cDNA was 1267 bp long (Fig. 1A). Analysis of the DNA sequence using the ORF finder program identified a 978-bp-long ORF. The predicated ORF encodes a protein of 326 amino acids with a molecular mass of 34.2 kDa (Fig. 1B). The theoretical isoelectric point of the protein was estimated to be 5.83. O-acetylserine (thiol) lyase is a pyridoxal phosphate-dependent enzyme, and a lysine residue at the N-terminal region of this protein is involved in binding this cofactor (Saito et al., 1993). This lysine residue and the sequence around it are also conserved in soybean OAS-TL (Fig. 1B). The BLASTX program and pairwise amino acid comparison of the soybean seed OAS-TL showed significant homology to OAS-TL from plants and bacteria. Soybean OAS-TL had 81% identity with Oryza sativa L., 80.5% identity with Arabidopsis thaliana (L) Heynh and 53.3% identity with E. coli OAS-TL (Fig. 2). A phylogenetic tree revealed that the OAS-TL isoforms could be divided into three major groups (chloroplastic, mitochondrial, and cytosolic) on the basis of their cellular location. Soybean OAS-TL was closely related to the cytosolic isoforms of OAS-TL from several other plant species (Fig. 2B). This prediction is consistent with our observation that the soybean OAS-TL lacks an amino terminal chloroplastic or mitochondrial transit peptide.

[FIGURES 1-2 OMITTED]

To determine the gene copy number of OAS-TL in soybean genome, we performed Southern blot analysis using genomic DNA that was digested with different restriction enzymes. The restricted DNA was transferred to a nylon membrane and probed with [sup.32]P-labeled OAS-TL cDNA. Under stringent hybridization conditions, we were able to detect more than one hybridizing band (Fig. 3) in the different restriction digestions. This observation suggests that the OAS-TL is probably encoded by a multigene family in soybean.

[FIGURE 3 OMITTED]

Functional Complementation of NK3 Cysteine E. coli Auxotroph by Soybean OAS-TL

To verify if the isolated cDNA clone codes for a functional OAS-TL, we expressed the soybean cDNA in E. coli NK3, a cysteine auxotroph. This mutant lacks the gene for OAS-TL and therefore cannot grow in medium without supplemental cysteine. We cloned the coding region of the soybean OAS-TL in a protein expression vector (pET28a) resulting in a plasmid pSCS10. Escherichia coli NK3, transformed with pSCS10, was able to grow on M9 minimal medium without cysteine (Fig. 4). The cysteine auxotroph and the mutant carrying the cloning vector, however, were unable to support the growth in the absence of cysteine (Fig. 4). These results confirm that the cDNA isolated from soybean seed cDNA library codes for a functional OAS-TL.

[FIGURE 4 OMITTED]

Temporal Expression of OAS-TL mRNA during Seed Development

For comparison of the OAS-TL gene transcription levels during seed development, we performed RT-PCR analysis using total RNA isolated from seeds at different developmental stages. Using primers designed to amplify the entire coding region of the OAS-TL, we were able to obtain 1.0-kb RT-PCR product (Fig. 5). The RT-PCR product, which was abundant during the early stages of seed development, declined perceptibly at the late stages of seed development (Fig. 5). To exclude the possibility that the decline in the OAS-TL mRNA at later stages of seed development was due to differences in the amount of total RNA used as template in RT-PCR reactions, we perfformed control reactions by amplifying a 700-bp 18S ribosomal RNA. As expected the abundance of the 18S ribosomal RNA RT-PCR products remained constant throughout the seed development (Fig. 5). The results from the RT-PCR analysis indicate that mRNA encoding the OAS-TL is abundant during the early stages and declines during the later stages of seed development.

[FIGURE 5 OMITTED]

Accumulation of OAS-TL Polypeptide during Seed Development

Proteins extracted from developing soybean seed when resolved by SDS-PAGE revealed the presence of prominent storage proteins (Fig. 6A). The 72- and the 70-kDa proteins are the [alpha]' and [alpha] subunits of [beta]-conglycinin. The 52-kDa [beta]-subunit of [beta]-conglycinin, which accumulates at late stages of seed development, was present only at very low concentration. The 37- and the 21-kDa abundant proteins represent the acidic and basic subunits of glycinin (Fig. 6A). To monitor the accumulation of the OAS-TL during seed development, Western blot analysis was performed with polyclonal antibodies raised against the purified soybean OAS-TL (Chronis and Krishnan, unpublished). The OAS-TL antibodies recognized a single 34-kDa protein from the total seed protein extract (Fig. 6B). The OAS-TL was detected throughout the seed development, but was present at relatively higher concentration during the early stages of seed development (Fig. 6B). This protein accumulation followed a similar trend as the RNA accumulation pattern.

[FIGURE 6 OMITTED]

OAS-TL Activity Declines during Seed Development

The activity of OAS-TL was measured at different ages of seed development (Fig. 7). The OAS-TL activity, which was measured spectrophotometrically, was readily detected in soybean seed extracts. The specific activity of the enzyme was highest during the earliest stage of seed development and declined eight fold during the last stage of seed development examined in this study (Fig. 7). The results from RT-PCR, Western blot analysis, and the enzyme activity assays all revealed similar temporal accumulation patterns.

[FIGURE 7 OMITTED]

DISCUSSION

Although the role of OAS-TL in cysteine biosynthesis has been extensively studied in several plants, to our knowledge this is the first report to identify a full-length cDNA clone of OAS-TL in soybean (GenBank accession no. AF452451). The amino acid sequence of the soybean OAS-TL cDNA shows significant homology to those of other plant species and bacteria. Soybean OAS-TL contains the conserved PXXSVKDR motif that is characteristic of cysteine synthase. The lysine residue in this conserved motif has been shown to bind the co-factor pyridoxal 5'-phosphate. The OAS-TL has been purified from several plant species including Arabidopsis thaliana. (Hesse and Altmann, 1995), spinach (Saito et al., 1992; Warrilow and Hawkesford, 1998), the green algae Chlamydomonas reinhardtii (Ravina et al., 1999), rice (Nakamura et al., 1999), Allium tuberosum Rottler ex Sprengel (Ikegami et al., 1993; Urano et al., 2000), Citrullus vulgaris Schrader (Ikegami et al., 1988a), and Brassica juncea (L.) Czernj. & Cosson(Ikegami et al., 1988b). The enzyme consists of two identical monomers and a tightly bound co-factor pyridoxal 5'-phosphate (Rolland et al., 1996). Two to four isoforms of the enzyme have been isolated in higher plants by chromatographic separations and cDNA isolations Ikegami et al., 1993; Kuske et al., 1994; Saito et al., 1992, 1993, 1994a, b; Warrilow and Hawkesford, 1998; Nakamura et al., 1999; Jost et at., 2000). The different isoforms of OAS-TL have been located in cytosol, plastids, and mitochondria. In A. thaliana, four genomic clones (oasA1, oasA2, oasB, and oasC) that encode OAS-TL have been identified and characterized. The oasA1, oasB, and oasC encode isoforms found in cytosol, the plastids, and the mitochondria, respectively. Based on the amino acid sequence homology, soybean OAS-TL appears to be related to the cytosolic isoforms.

The OAS-TL plays an important role in linking sulfur and nitrogen assimilatory pathways and controlling the flux between these two pathways (Leustek and Saito, 1999; Leustek et al., 2000). Consequently, cysteine synthesis depends on the availability of sulfur, OAS, and the activity of OAS-TL. The accumulation of soybean seed storage proteins is regulated by sulfur and nitrogen availability. Under excess nitrogen supply, the accumulation of the [beta]-subunit of [beta]-conglycinin is enhanced, While that of glycinin is inhibited (Gayler and Sykes, 1985; Pack et al., 1997). Kim et al. (1999) have shown that the promoter of the [beta]-subunit of [beta]-conglycinin is up-regulated by sulfur deficiency and downregulated by nitrogen deficiency. Further, they have shown that OAS accumulates in soybean cotyledons that were cultured under sulfur deficiency. This study clearly establishes the pivotal role of OAS in regulating the accumulation of the soybean seed storage proteins. Since OAS-TL uses OAS as a substrate it should be interesting to examine if nitrogen and sulfur deficiency also influence its activity in soybean.

The activity of ATP sulfurylase, an enzyme that catalyzes the adenylation of sulfate, has been investigated in developing soybean seeds (Sexton and Shibles, 1999). It was reported that the ATP sulfurylase activity was highest in seeds harvested 15 d after the R5 stage (about 1600 nmol ATP g fresh [wt.sup-1] [min.sup.-1]) and reached low levels (about 250 nmol ATP g fresh [wt.sup-1] [min.sup.-1]) at the R7 stage. We have observed similar changes in the OAS-TL specific activity. The RT-PCR results indicated hat OAS-TL mRNA was barely present in mature seeds and this led to the observed decline in OAS-TL activity during the later stages of seed development. It remains to be seen if a similar decline in ATP sulfurylase mRNA also occurs during seed maturation. The decline in the activity of two enzymes involved in the biosynthesis of cysteine may explain the low content of sulfur-rich amino acids in soybean seed proteins. Because the bulk of seed storage proteins are synthesized during the mid-stage of seed development, it would be desirable to have sufficient supply of cysteine during this period. However, the decline in the activity of OAS-TL and ATP sulfurylase during this period indicates that the supply of sulfur-amino acids may be limiting. The limitation on cysteine can be overcome by manipulating the expression levels of enzymes involved in cysteine biosynthetic pathway.

The cysteine biosynthetic pathway is tightly regulated at several levels (Leustek and Saito, 1999; Leustek et al., 20000; Noji and Saito, 2002). The end product of sulfur assimilation, cysteine, is an allosteric inhibitor of the cytosolic form of serine acetyltransferase (SAT; EC 2.3.1.30). Serine acetyltransferase catalyses the formation of OAS from acetyl-CoA and serine. The OAS-TL activity is also regulated by its interaction with SAT (Bogdanova and Hell, 1997; Droux et al., 1998). OAS-TL and SAT form an enzyme complex through specific protein-protein interactions. In the bound form, SAT shows positive cooperativity, meaning higher affinity for its substrates. On the other hand, OAS-TL is completely inactivated in the bound form. OAS triggers the dissociation of the complex, and sulfide counteracts the action of OAS (Bogdanova and Hell, 1997; Droux et al., 1998). A lag in sulfide production will result in accumulation of OAS, which will slow its own synthesis by promoting the dissociation of the complex. Alternatively, the accumulation of sulfide will act as positive regulator in the association of SAT and OAS-TL thereby speeding the formation of OAS. Because the level of OAS influences the composition of soybean seed storage proteins (Kim et al., 1999), it will be important to clone and characterize soybean SAT, the enzyme responsible for the generation of OAS.

Abbreviations: OAS-TL, O-acetylserine (thiol) lyase; ORF, open reading frame; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction, SAT, serine acetyltransferase.

ACKNOWLEDGMENTS

We thank Dr. Kazuki Saito (Chiba University, Chiba, Japan) for providing us with the NK3 E. coil mutant. The authors would like to thank Drs. Larry Darrah and Jerry White for critical reading of the manuscript.

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Demosthenis Chronis and Hari B. Krishnan *

D. Chronis, Dep. of Agronomy, Univ. of Missouri, Columbia, MO 65211; H.B. Krishnan, USDA-ARS, Plant Genetics Research Unit, Univ. of Missouri, Columbia, MO 65211. Received 20 Nov. 2002. * Corresponding author (KrishnanH@missouri.edu).
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Title Annotation:Genomics, Molecular Genetics & Biotechnology
Author:Chronis, Demosthenis; Krishnan, Hari B.
Publication:Crop Science
Date:Sep 1, 2003
Words:5267
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