Post-emergence application of glufosinate on maize hybrids containing the phosphinothricin acetyltransferase gene (pat)

Several maize hybrids that present the phosphinothricin acetyltransferase gene (pat) are available in the market. However, these hybrids have different resistance levels to glufosinate herbicides. The objective of the present work was to evaluate the resistance of maize hybrids containing the pat gene (as a selection marker) to glufosinate. Field experiments were conducted in two sites in the 2016/2017 crop season, using a randomized block design with a 2×7 factorial arrangement and four replications. The treatments consisted of two glufosinate rates (0 and 500 g ha-1) and seven maize hybrids, six containing the pat gene as a selection marker (Herculex®, Agrisure-TL®, Herculex Yieldgard®, Leptra®, Viptera-3®, and Power-Core®) and one without the pat gene (VT PRO®). Two field experiments were conducted in different sites. The analyzed variables were: ammonia accumulation, electron transport rate (ETR), percentage of injuries, 100-grain weight, and grain yield. The glufosinate-susceptible maize hybrid presented higher ammonia accumulations, lower ETR, and high percentage of injuries (100%), which caused total loss of grain production. Considering the evaluated glufosinate-resistant maize hybrids, Viptera-3 and Agrisure-TL presented the highest ammonia accumulations and percentages of injuries, and lower ETR than the other hybrids. The grain yield of glufosinate-resistant maize hybrids was not reduced due to the application of the 500 g ha-1 of glufosinate. Thus, glufosinate-resistant maize hybrids containing the pat gene are resistant to the application of 500 g ha-1 of glufosinate, and this practice can be recommended for maize crops.

Discovery and reduction of nonspecific activities of the major herbicide-resistance gene BAR

Herbicide resistance is a major trait of genetically modified (GM) crops. Currently, resistance to phosphinothricin (also known as glufosinate) is the second most widespread genetically engineered herbicide-resistance trait in crops after glyphosate resistance1,2. Resistance to phosphinothricin in plants is achieved by transgenic expression of the bialaphos resistance (BAR) or phosphinothricin acetyltransferase (PAT) genes, which were initially isolated from the natural herbicide bialaphos-producing soil bacteria Streptomyces hygroscopicus and S. viridochromogenes, respectively3,4. Mechanistically, BAR and PAT encode phosphinothricin acetyltransferase, which transfers an acetyl group from acetyl coenzyme A (acetyl-CoA) to the α-NH2 group of phosphinothricin, resulting in herbicide inactivation1. Although early in vitro enzyme assays showed that recombinant BAR and PAT exhibit substrate preference toward phosphinothricin over the 20 proteinogenic amino acids1, whether transgenic expression of BAR and PAT affects plant endogenous metabolism in vivo was not known. Combining metabolomics, plant genetics, and biochemical approaches, we show that transgenic BAR indeed converts two plant endogenous amino acids, aminoadipate and tryptophan, to their respective N-acetylated products in several plant species examined. We report the crystal structures of BAR, and further delineate structural basis for its substrate selectivity and catalytic mechanism. Through structure-guided protein engineering, we generated several BAR variants that display significantly reduced nonspecific activities compared to its wild-type counterpart. Our results demonstrate that transgenic expression of enzymes as a common strategy in modern biotechnology may render unintended metabolic consequences arisen from enzyme promiscuity. Understanding of such phenomena at the mechanistic level will facilitate better design of maximally insulated systems featuring heterologously expressed enzymes.