An in vivo internal deletion in the N-terminus region of Arabidopsis cystathionine gamma-synthase results in CGS expression that is insensitive to methionine.

Cystathionine gamma-synthase (CGS), the first enzyme of methionine biosynthesis in higher plants, plays an important role in the biosynthesis pathway and in regulating methionine metabolism in plant cells. In response to methionine, the expression of this enzyme is regulated via amino acid sequences located in its N-terminal. Here, using reverse transcription PCR and ribonuclease protection analysis, we demonstrate that, in addition to the full-length CGS transcript, a deleted form exists in Arabidopsis. The deleted transcript of CGS that lacks 90 or 87 nt located internally in the regulatory N-terminal region of CGS maintains the reading frame of the protein. Its association with polyribosomes indicates that this deleted form of CGS is translated. In order to study the function of this deleted form of CGS, we overexpressed it in transgenic tobacco plants. We found that the transgenic plants engineered to express only the deleted form of CGS accumulated methionine to a much higher level than those that expressed the full-length CGS. Furthermore, in vitro feeding experiments revealed that the deleted form of CGS is not subject to feedback regulation by methionine, as reported for the full-length transcript. Therefore, although most likely produced from the full-length CGS, the transcript of the deleted form is insensitive to methionine application and its expression may be important for maintaining methionine metabolism even in the presence of a high level of methionine.

[1]  S. Naito,et al.  Nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in the CGS1 gene of Arabidopsis. , 2005, Genes & development.

[2]  R. Amir,et al.  The expression level of threonine synthase and cystathionine-γ-synthase is influenced by the level of both threonine and methionine in Arabidopsis plants , 2005, Transgenic Research.

[3]  Jiyeon Lee,et al.  Methionine and threonine synthesis are limited by homoserine availability and not the activity of homoserine kinase in Arabidopsis thaliana. , 2005, The Plant journal : for cell and molecular biology.

[4]  S. Naito,et al.  Autoregulation of the gene for cystathionine gamma-synthase in Arabidopsis: post-transcriptional regulation induced by S-adenosylmethionine. , 2004, Biochemical Society transactions.

[5]  H. Hesse,et al.  Current understanding of the regulation of methionine biosynthesis in plants. , 2004, Journal of experimental botany.

[6]  G. Schuster,et al.  Altering the 3′ UTR endonucleolytic cleavage site of a Chlamydomonas chloroplast mRNA affects 3′-end maturation in vitro but not in vivo , 1999, Plant Molecular Biology.

[7]  S. Naito,et al.  Decay kinetics of autogenously regulated CGS1 mRNA that codes for cystathionine gamma-synthase in Arabidopsis thaliana. , 2003, Plant & cell physiology.

[8]  S. Naito,et al.  S-adenosyl-l-methionine is an effector in the posttranscriptional autoregulation of the cystathionine γ-synthase gene in Arabidopsis , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[9]  H. Hesse,et al.  Molecular aspects of methionine biosynthesis. , 2003, Trends in plant science.

[10]  G. Schuster,et al.  HCF152, an Arabidopsis RNA Binding Pentatricopeptide Repeat Protein Involved in the Processing of Chloroplast psbB-psbT-psbH-petB-petD RNAs Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010397. , 2003, The Plant Cell Online.

[11]  H. Hesse,et al.  Functional Analysis of Cystathionine γ-Synthase in Genetically Engineered Potato Plants1 , 2003, Plant Physiology.

[12]  S. Naito,et al.  Identification of a Short Highly Conserved Amino Acid Sequence as the Functional Region Required for Posttranscriptional Autoregulation of the Cystathionine γ-Synthase Gene in Arabidopsis * , 2002, The Journal of Biological Chemistry.

[13]  G. Galili,et al.  Cystathionine gamma-synthase and threonine synthase operate in concert to regulate carbon flow towards methionine in plants. , 2002, Trends in plant science.

[14]  Y. Hacham,et al.  The N-Terminal Region of Arabidopsis Cystathionine γ-Synthase Plays an Important Regulatory Role in Methionine Metabolism1 , 2002, Plant Physiology.

[15]  W. Boerjan,et al.  Constitutive overexpression of cystathionine gamma-synthase in Arabidopsis leads to accumulation of soluble methionine and S-methylmethionine. , 2002, Plant physiology.

[16]  U. Roessner,et al.  Antisense inhibition of threonine synthase leads to high methionine content in transgenic potato plants. , 2001, Plant physiology.

[17]  S. Naito,et al.  Mutation in the threonine synthase gene results in an over-accumulation of soluble methionine in Arabidopsis. , 2000, Plant physiology.

[18]  S. Ravanel,et al.  METHIONINE BIOSYNTHESIS IN PLANTS: BIOCHEMICAL AND REGULATORY ASPECTS , 2000 .

[19]  Satoshi Naito,et al.  Evidence for autoregulation of cystathionine γ-synthase mRNA stability in Arabidopsis , 1999 .

[20]  J. Kim,et al.  Evidence for autoregulation of cystathionine gamma-synthase mRNA stability in Arabidopsis. , 1999, Science.

[21]  S. Ravanel,et al.  The specific features of methionine biosynthesis and metabolism in plants. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[22]  S. Ravanel,et al.  Cystathionine gamma-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli. , 1998, The Biochemical journal.

[23]  T. Shinada,et al.  Biosynthesis of Phytosiderophores, Mugineic Acids, Associated with Methionine Cycling (*) , 1995, The Journal of Biological Chemistry.

[24]  G. Galili,et al.  Increased lysine synthesis in tobacco plants that express high levels of bacterial dihydrodipicolinate synthase in their chloroplasts , 1992 .

[25]  Mudd Sh,et al.  The S-Methylmethionine Cycle in Lemna paucicostata , 1990 .

[26]  Thomas J. White,et al.  PCR protocols: a guide to methods and applications. , 1990 .

[27]  A. Datko,et al.  The S-Methylmethionine Cycle in Lemna paucicostata. , 1990, Plant physiology.

[28]  A. Barkan.  Tissue-dependent plastid RNA splicing in maize: transcripts from four plastid genes are predominantly unspliced in leaf meristems and roots. , 1989, The Plant cell.

[29]  J. Fry,et al.  A simple and general method for transferring genes into plants. , 1985, Science.