HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text Free Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (8) Citing Articles via Google Scholar Google Scholar Articles by Chronis, D. Articles by Krishnan, H. B. Search for Related Content PubMed Articles by Chronis, D. Articles by Krishnan, H. B. Agricola Articles by Chronis, D. Articles by Krishnan, H. B. Related Collections Plant Nutrition Seed Physiology

Sulfur Assimilation in Soybean

Molecular Cloning and Characterization of O-Acetylserine (Thiol) Lyase (Cysteine Synthase)

^a Dep. of Agronomy, Univ. of Missouri, Columbia, MO 65211
^b USDA-ARS, Plant Genetics Research Unit, Univ. of Missouri, Columbia, MO 65211

View larger version (65K): [in a new window]   Fig. 1. A partial restriction map of soybean cDNA encoding the OAS-TL. The long arrow indicates the location of the open-reading frame (ORF). (B). Nucleotide sequence and deduced amino acid sequence of OAS-TL cDNA from soybean seed. The sequenced region covers 1267 nucleotides. The ORF for OAS-TL begins at position 82 and ends at position 1059 encoding a 34.2-kDa protein. The lysine residue that binds to pyridoxal 5'-phosphate is circled. The nucleotide sequence of OAS-TL cDNA from soybean appears in the GenBank database as accession No. AF452451.

View larger version (90K): [in a new window]   Fig. 2. (A). Multiple alignment of the deduced amino acid sequence of OAS-TL. The sequences from rice (Accession No. Q9XEA8), Arabidopsis (Accession No. NP_193224), and E. coli (Accession No. P11096] are aligned with that of the soybean sequences from this study (Accession No. AF452451). Positions of amino acid identity are shaded black and similar residues are shaded in gray. (B) Phylogenic tree of OAS-Tl. The phylogenic tree was constructed using the University of California database. Cytosolic isoforms: Glycine max (Accession No. AF452451), Arabidopsis thaliana (Accession No. NP_193224), Solanum tuberosum (Accession No. BAB20861), Brassica juncea (Accession No. O23733), Spinacea oleracea (Accession No. Q00834), Citrulus lanatus (Accession No. Q43317), Oryza sativa (Accession No. Q9XEA8), Zea mays (Accession No. P80608), Allium tuberosum (Accession No. BAA93051), and Triticum aestivum (Accession No. P38076). Chloroplastic isoforms: Capsicum annuum (Accession No. P31300), Spinacea oleracea (Accession No. D14722), Nicotiana tabacum (Accession No. AJ299249), Solanum tuberosum (Accession No. O81155), and Arabidopsis thaliana (Accession No. S48695). Mitochondrial isoform: Arabidopsis thaliana (Accession No. X81973). Bacterial OAS-TL: Escherichia coli (Accession No. P11096), Nostocaceae (Accession No. NC_003272), Thermosynechococcus elongates (Accession No. NC_004113), Synechocystis (Accession No. P73410), and Mesorhizobium loti (Accession No. NC_002678).

View larger version (40K): [in a new window]   Fig. 3. Southern blot analysis of soybean genomic DNA. Ten micrograms of soybean genomic DNA was restricted with BamHI (lane 1), EcoRI (lane 2), and HindIII (lane 3) and resolved on a 0.8% (w/v) agarose gel. The gel was blotted to Hybond N+ membrane followed by hybridization with 32P-labeled soybean seed OAS-TL cDNA insert. The positions of the Lambda HindIII molecular weight markers are shown at the left side of the figure.

View larger version (63K): [in a new window]   Fig. 4. Functional complementation of Cys- E. coli NK3 by transformation with the expression vector carrying soybean OAS-TL cDNA clone. The E. coli cysteine-auxotroph was transformed with pSCS10 and was streaked on M9 minimal agar plates with 0.5 mM cysteine (right plate) or without cysteine (left plate). The empty vector pET28a was used as a negative control.

View larger version (32K): [in a new window]   Fig. 5. Reverse transcriptase (RT)-PCR detection of OAS-TL mRNA in developing soybean seeds. Total RNA isolated from soybean seeds harvested at 7-d intervals from 5 d after R5 stage (lanes 1–6) was used as a template for RT-PCR. The 18S ribosomal mRNA was used as quantitative control. Sizes of the molecular weight markers are indicated on the right side of the figure.

View larger version (43K): [in a new window]   Fig. 6. Accumulation of OAS-TL during soybean seed development. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein profiles of developing soybean seeds. Total seed proteins isolated from six different developmental stages (lanes 1–6) were resolved on a 10% (w/v) SDS-polyacrylamide gel and stained with Coomassie brilliant blue. (B). Western blot analysis. Total protein from developing soybean seeds was resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies raised against the soybean OAS-TL. Note that the antibody specifically recognizes a 34-kDa protein from the soybean seed extracts. The numbers in kilodaltons shown at the sides of the figures represent Bio-Rad (Richmond, CA) protein molecular weight markers (phosphorylase b, 97 400; bovine serum albumin, 66 200; ovalbumin, 45 000; carbonic anhydrase, 31 000; soybean trypsin inhibitor, 21 500; lysozyme, 14 400).

View larger version (23K): [in a new window]   Fig. 7. OAS-TL activity in soybean seeds. Seed samples from nodes 10 and 11 were collected at weekly intervals starting from R5 and cysteine synthase activity was measured using crude seed extracts. Formation of cysteine was determined with an OAS-ninhydrin assay. Bars represent the standard error of the mean.