Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination, callus and shoot formation.
A novel acetyltransferase (Mpr1) found in Saccharomyces cerevisiae (strain Sigma1278b) has been shown to specifically detoxify a proline analog, l-azetidine-2-carboxylic acid (A2C) in yeast cells [M. Shichiri et al. (2001) J Biol Chem 276: 41998-42002]. We investigated whether the yeast MPR1 gene would function similarly in a plant system and if its expression could confer resistance to proline analogs. The MPR1 gene coding sequence driven by two different constitutive promoters, with or without the 5'- and 3'-noncoding sequence from the MPR1 gene adjacent to the conventional NOS terminator, was transformed into tobacco ( Nicotiana tabacum L. cv. Xanthi) plants via Agrobacterium tumefaciens infection. The presence of the yeast 5'- and 3'-noncoding sequences appeared to increase the likelihood of MPR1 gene expression in the transgenic plants. The kanamycin-selected transgenic plants with a high level of Mpr1 activity grew normally, and their progeny expressed acetyltransferase activity that could utilize A2C, azetidine-3-carboxylic acid and 4-hydroxy- l-proline as substrates. Resistance to A2C, but not to the other two analogs, was exhibited during leaf tissue culture and seed germination. The A2C toxicity to the wild-type plants was reversed by the addition of proline, suggesting that A2C acts as a proline analog. Our studies confirm that MPR1 can function in a similar fashion in tobacco as in yeast to detoxify the toxic proline analog A2C, so it could potentially be used as a new selectable marker for plant transformation. However, our attempts to utilize MPR1 as an efficient selectable marker gene for the A. tumefaciens-mediated transformation of tobacco were unsuccessful.
Zhang
X-H
XH
Department of Crop Sciences, E.R. Madigan Laboratory, University of Illinois, 1201 W Gregory Drive, Urbana, IL 61801, USA.
Takagi
H
H
Widholm
J M
JM
D001383:Azetidinecarboxylic Acid / Q000378:metabolism / Q000633:toxicity*; D001483:Base Sequence; D046508:Culture Techniques; D004352:Drug Resistance, Microbial / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D005822:Genetic Vectors; D018525:Germination; D030821:Plants, Genetically Modified / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology; D012639:Seeds / Q000254:growth & development*; D014026:Nicotiana / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic
D029701:Saccharomyces cerevisiae Proteins; D001383:Azetidinecarboxylic Acid