Selectable Tolerance to Herbicides by Mutated Acetolactate Synthase Genes Integrated into the Chloroplast Genome of Tobacco  

Abstract

Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicideresistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3#-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.

Plastid Biotechnology: Food, Fuel, and Medicine for the 21st Century

“…By now, resistances to the same set of herbicides have been obtained by expression of transgenes in plastids. Examples include resistance to glyphosate (Ye et al, 2001), Bialaphos/ Liberty (Iamtham and Day, 2000;Lutz et al, 2001), isoxaflutole (Dufourmantel et al, 2007), and sulfonylurea herbicides (Shimizu et al, 2008). If these plastid transgenes are incorporated in commercial crops, plastid localization provides an efficient containment tool Figure 6.…”

Transplastomic plants for innovations in agriculture. A review

“…Herbicides can also be used as selective agents (Table 1); therefore, much effort has been devoted to develop transplastomic plants with resistance to different herbicides such as glyphosate (Daniell et al 1998;Ye et al 2001Ye et al , 2003Chin et al 2003;Roudsari et al 2009), phosphinothricin/ glufosinate ammonium (Iamtham and Day 2000;Lutz et al 2001;Kang et al 2003;Ye et al 2003), sulcotrione (Falk et al 2005), isoxaflutole/diketonitrile (Dufourmantel et al 2007), chlorophenylthio-triethylamine (CPTA) (Wurbs et al 2007), pyrimidinylcarboxylate, imidazolinone and sulfonylurea (Shimizu et al 2008), and paraquat (methyl-viologen) (Le Poage et al 2011;Chen et al 2014). Herbicide resistance is achieved (1) by the insertion of a bacterial marker gene (such as bar) encoding an enzyme that inactivates the herbicide (phosphinothricin/glufosinate ammonium-Iamthan and Day 2000; Lutz et al 2001Lutz et al , 2006, (2) by overexpression of the genes of plastidial metabolic enzymes that are the targets of herbicides (e.g., EPSPS: glyphosate- Daniell et al 1998; hppd: sulcotrione and the isoxaflutole derivative, diketonitrile -Falk et al 2005 andDufourmantel et al 2007, respectively), (3) by expression of the genes of mutant, herbicide-resistant plant enzymes (CP4: glyphosate- Ye et al 2001Ye et al , 2003Roudsari et al 2009; mALS: pyrimidinylcarboxylate, imidazolinone, and sulfonylurea- Shimizu et al 2008), or (4) by expression of enzyme genes involved in antioxidant defense, minimizing this way the metabolic impact of the herbicides via the generation of ROS (DHAR, GST, gor: paraquat/methyl-viologen-Le MnSOD: paraquat/methyl-viologen-Poage et al 2011). For instance, glyphosate is a competitive inhibitor of one enzyme of the plastid aromatic amino acid biosynthesis pathway, namely 5-enolpyruvylshikimate-3-phosphate (EPSPS).…”

Growth of Transplastomic Cells Expressing d-Amino Acid Oxidase in Chloroplasts Is Tolerant to d-Alanine and Inhibited by d-Valine

“…Reverse genetics allows site-directed changes to be introduced into the important set of genes present in plastids to study and ultimately improve their function (Whitney et al, 2011;Day, 2012). Positive conditional selectable marker genes allow transgenic cells to divide in the presence of chemicals such as antibiotics or herbicides that inhibit wild-type untransformed cells and underpin the development of herbicide-resistant transplastomic crops (Daniell et al, 1998(Daniell et al, , 2009Iamtham and Day, 2000;Lutz et al, 2001;Ye et al, 2001;Dufourmantel et al, 2007;Shimizu et al, 2008). Negative conditional selectable marker genes inhibit the proliferation of transgenic cells when exposed to compounds that have a limited impact on the viability of wild-type cells (Miki and McHugh, 2004).…”