Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination, callus and shoot formation

Abstract

A novel acetyltransferase (Mpr1) found in Saccharomyces cerevisiae (strain Σ1278b) 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.