Submitted to: Planta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/10/2003
Publication Date: 10/30/2003
Citation: Chronis, D., Krishnan, H.B. 2003. Sulfur assimilation in soybean (glycine max [l.] merr.): molecular cloning and characterization of a cytosolic isoform of serine acetyltransferase. Planta. V.218:417-426 Interpretive Summary: Soybean is a rich source of protein. Unfortunately, soybean proteins contain low amounts of two important amino acids, methionine and cysteine, that are vital for optimal growth of humans and animals. Therefore, attempts are being made to increase the amount of these two amino acids in soybean proteins. This study reports the cloning and characterization of a key enzyme that is involved in the production of cysteine. Our results demonstrate that this enzyme is expressed at very low levels in developing seeds. Since this enzyme plays a major role in the synthesis of cysteine, it is desirable to elevate the expression of this protein during soybean seed development. The information obtained from this basic study will help biotechnologists to genetically manipulate the sulfur-assimilatory enzyme expression levels so that we can improve the overall quality of soybean seed proteins. Superior quality soy proteins can be utilized to meet the nutritional requirements of the multitude of malnourished people around the world.
Technical Abstract: A full-length cDNA clone encoding a cytosolic isoform of serine acetyltransferase (SATase) (EC 126.96.36.199) 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 mutant in minimal medium without cysteine. Expression of soybean SATase in E. coli resulted in the production of a 34-kDa protein which 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 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.