Vacuolar Glyphosate-Sequestration Correlates with Glyphosate Resistance in Ryegrass (Lolium spp.) from Australia, South America, and Europe: A 31 P NMR Investigation Xia Ge, † D. Andre ́ d’Avignon, † Joseph J. H. Ackerman,* ,†,‡,# Alberto Collavo, § Maurizio Sattin, § Elizabeth L. Ostrander, ⊥ Erin L. Hall, ⊥ R. Douglas Sammons, ⊥ and Christopher Preston ⊗ † Departments of Chemistry, ‡ Internal Medicine, and # Radiology Washington University, 1 Brookings Drive, St. Louis, Missouri 63130, United States § Institute of Agro-environmental and Forest Biology, Viale dell’Universita ̀ 16, 35020 Legnaro (PD), Italy ⊥ Monsanto Company, 800 Lindbergh Boulevard, St. Louis, Missouri 63167, United States ⊗ School of Agriculture, Food and Wine, The University of Adelaide, PMB 1, Glen Osmond, SA 5064, Australia * S Supporting Information ABSTRACT: Lolium spp., ryegrass, variants from Australia, Brazil, Chile, and Italy showing differing levels of glyphosate resistance were examined by 31 P NMR. Extents of glyphosate (i) resistance (LD 50 ), (ii) inhibition of 5-enopyruvyl-shikimate- 3-phosphate synthase (EPSPS) activity (IC 50 ), and (iii) translocation were quantified for glyphosate-resistant (GR) and glyphosate-sensitive (GS) Lolium multiflorum Lam. variants from Chile and Brazil. For comparison, LD 50 and IC 50 data for Lolium rigidum Gaudin variants from Italy were also analyzed. All variants showed similar cellular uptake of glyphosate by 31 P NMR. All GR variants showed glyphosate sequestration within the cell vacuole, whereas there was minimal or no vacuole sequestration in the GS variants. The extent of vacuole sequestration correlated qualitatively with the level of resistance. Previous 31 P NMR studies of horseweed (Conyza canadensis (L.) Cronquist) revealed that glyphosate sequestration imparted glyphosate resistance. Data presented herein suggest that glyphosate vacuolar sequestration is strongly contributing, if not the major contributing, resistance mechanism in ryegrass as well. KEYWORDS: glyphosate, glyphosate resistance, in vivo 31 P NMR, Lolium spp., glyphosate tonoplast transporter ■ INTRODUCTION Glyphosate is the world’s most important and widely used herbicide. 1 The introduction of glyphosate-resistant crops in 1996 extended its global use. 2 Glyphosate inhibits the plastidic enzyme 5-enopyruvyl-shikimate-3-phosphate synthase (EPSPS) located in cell chloroplast. 3 EPSPS is critical to the shikimate biosynthesis pathway, which is the source for up to 30% of the complex carbon-containing moieties in higher plants. 4 Inhibi- tion of EPSPS curtails this critical biosynthetic pathway and ultimately results in plant death. It follows that for glyphosate to be effective, it must be taken up by the plant cell and must further enter the chloroplast to inhibit plastidic EPSPS. Glyphosate is translocated symplastically by the phloem from photosynthetically active source leaves to highly sensitive sink tissues, leading to whole plant death. 5-7 After over 35 years of continuous glyphosate usage, selective weed species have evolved glyphosate-resistant (GR) popula- tions. 8,9 Today 21 GR weed species have been discovered worldwide. 10 Common agricultural delivery of glyphosate is at the millimolar level with a fraction taken up and a smaller frac- tion of this amount translocated to sink-tissue chloroplasts. 11 Although chloroplast glyphosate concentrations at the sub- micromolar level are sufficient to effectively inhibit EPSPS function, 12-14 anything that compromises chloroplast glyphos- ate delivery could play an important role in establishing herbi- cide resistance. 7,15 Ryegrass species are economically important in several re- gions of the world. 16 There are three well-recognized cross- pollinated species within the Lolium genus: L. rigidum Gaudin (Stiff darnel, Wimmera ryegrass), L. multiflorum Lam. (Italian ryegrass), and L. perenne L. (perennial ryegrass). These three Lolium species freely cross-pollinate, and the hybrids are highly fertile. 17 Glyphosate-resistant L. rigidum was discovered in Australia in 1996. 11,18,19 Today, L. rigidum ranks in the top 10 most important herbicide-resistant species. Both EPSPS muta- tion (target-site) and reduced translocation (non-target-site) have been suggested to play a role, but non-target-site resis- tance may be the major GR mechanism in rigid ryegrass. 20,21 Millimolar concentrations of glyphosate in the plant cell can be readily monitored by 31 P NMR in vivo, 22,23 a spectroscopic technique that can be advantageously combined with a pulse- chase protocol to quantify glyphosate partitioning by plants. 22 In this protocol, perfused plant tissue is exposed to herbicide in the perfusate for a fixed time period (the pulse phase). Switch- ing to a glyphosate-free perfusion medium at the conclusion of the pulse phase washes unincorporated herbicide from the plant surface and apoplast (the wash phase). Following the Received: August 30, 2011 Revised: December 15, 2011 Accepted: January 4, 2012 Published: January 4, 2012 Article pubs.acs.org/JAFC © 2012 American Chemical Society 1243 dx.doi.org/10.1021/jf203472s | J. Agric.Food Chem. 2012, 60, 1243-1250