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Sulfur assimilation in soybean (Glycine max [L.] Merr.): molecular cloning and characterization of a cytosolic isoform of serine acetyltransferase

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Abstract

A full-length cDNA clone encoding a cytosolic isoform of serine acetyltransferase (SATase; EC 2.3.1.30) was isolated by screening a soybean seedling cDNA library with a 32P-labeled expressed sequence tag. Nucleotide sequence analysis of the isolated cDNA revealed a single open-reading frame of 858 base pairs encoding a 30-kDa polypeptide. The deduced amino acid sequence of soybean SATase revealed significant homology with other plant SATases. Analysis of genomic DNA by Southern blotting indicated that SATase is encoded by a small gene family. The authenticity of the isolated SATase cDNA was confirmed by the expression of the cDNA in an Escherichia coli cysteine-auxotrophic mutant resulting in the growth of the mutant in minimal medium without cysteine. Expression of soybean SATase in E. coli resulted in the production of a 34-kDa protein that was subsequently purified by nickel-affinity column chromatography. The purified protein exhibited SATase activity, indicating that the E. coli-expressed protein is a functionally active SATase. The recombinant soybean SATase was inhibited by l-cysteine, the end product of the cysteine biosynthetic pathway. Antibodies raised against the recombinant soybean SATase cross-reacted with a 34-kDa protein from Arabidopsis leaves, but failed to detect any proteins from soybean leaves and seeds. Reverse transcriptase–polymerase chain reaction analysis indicated that SATase mRNA was expressed at low levels during soybean seed development. In comparison to Arabidopsis leaves, the SATase activity was several-fold lower in soybean leaves and seeds, suggesting that SATase is a low-abundance enzyme.

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Abbreviations

EST:

expressed sequence tag

IPTG:

isopropyl-beta-d-thiogalactopyranoside

SATase:

serine acetyltransferase

OAS-TL:

O-acetylserine (thiol)-lyase (cysteine synthase)

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Acknowledgements

We thank Dr. Thomas Leustek (Rutgers University, NJ, USA) for providing us with the Arabidopsis SATase antibody and Dr. Joe Polacco (University of Missouri, MO, USA) for providing us with the soybean seedling cDNA library. We also thank Dr. Larry Darrah and John Bennett for critically reading the manuscript. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may be suitable.

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  1. Plant Genetics Research Unit, USDA–Agricultural Research Service and Department of Agronomy, University of Missouri, Columbia, MO 65211, USA

    Hari B. Krishnan

  2. Department of Agronomy, University of Missouri, Columbia, MO 65211, USA

    Demosthenis Chronis

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  1. Demosthenis Chronis
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  2. Hari B. Krishnan
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Correspondence to Hari B. Krishnan.

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Chronis, D., Krishnan, H.B. Sulfur assimilation in soybean (Glycine max [L.] Merr.): molecular cloning and characterization of a cytosolic isoform of serine acetyltransferase. Planta 218, 417–426 (2004). https://doi.org/10.1007/s00425-003-1123-3

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  • DOI: https://doi.org/10.1007/s00425-003-1123-3

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