Focus on Weed Control: In Vivo 31P-Nuclear Magnetic Resonance Studies of Glyphosate Uptake,Vacuolar Sequestration,and Tonoplast Pump Activity in Glyphosate-Resistant Horseweed

Horseweed (Conyza canadensis) is considered a significant glyphosate-resistant (GR) weed in agriculture, spreading to 21 states in the United States and now found globally on five continents. This laboratory previously reported rapid vacuolar sequestration of glyphosate as the mechanism of resistance in GR horseweed. The observation of vacuole sequestration is consistent with the existence of a tonoplast-bound transporter. ³¹P-Nuclear magnetic resonance experiments performed in vivo with GR horseweed leaf tissue show that glyphosate entry into the plant cell (cytosolic compartment) is (1) first order in extracellular glyphosate concentration, independent of pH and dependent upon ATP; (2) competitively inhibited by alternative substrates (aminomethyl phosphonate AMPA] and N-methyl glyphosate NMG]), which themselves enter the plant cell; and (3) blocked by vanadate, a known inhibitor/blocker of ATP-dependent transporters. Vacuole sequestration of glyphosate is (1) first order in cytosolic glyphosate concentration and dependent upon ATP; (2) competitively inhibited by alternative substrates (AMPA and NMG), which themselves enter the plant vacuole; and (3) saturable. ³¹P-Nuclear magnetic resonance findings with GR horseweed are consistent with the active transport of glyphosate and alternative substrates (AMPA and NMG) across the plasma membrane and tonoplast in a manner characteristic of ATP-binding cassette transporters, similar to those that have been identified in mammalian cells.Glyphosate is arguably the world’s most important herbicide (Duke and Powles, 2008). Environmental factors affecting its uptake and translocation in higher plants have been widely studied (Kells and Rieck, 1979; Coupland, 1983; Devine et al., 1983; Masiunas and Weller, 1988; Zhou et al., 2007). Notably, the role of light is important for effective uptake and translocation, suggesting that metabolic energy plays a role in this process (Simarmata et al., 2003; Shaner et al., 2005). Death of the whole plant requires effective glyphosate translocation from source to sink tissue, a process requiring ATP to maintain Suc gradients, which drive phloem movement (Bromilow et al., 1990; Dill et al., 2010).Weed species have developed glyphosate-resistant (GR) biotypes during the past decade (Heap, 2014). This has spurred interest in factors that may contribute to resistant attribute(s) as well as methods that can be used to screen plants for herbicide toxicity (Shaner, 2010). Resistance mechanisms have been reported for horseweed (Conyza canadensis; Feng et al., 2004; Zelaya et al., 2004; Ge et al., 2010, 2011), Palmer amaranth (Amaranthus palmeri; Gaines et al., 2010), and ryegrass (Lolium rigidum and Lolium multiflorum; Powles et al., 1998; Perez et al., 2004; Ge et al., 2012).Since glyphosate is foliar applied, glyphosate toxicity involves a multistep delivery process. Glyphosate must traverse the nonliving structures of the leaf cuticle and the cell walls of the epidermis, apoplast, and mesophyll prior to accessing the phloem for transport to sink tissues (Bromilow et al., 1990; Bromilow and Chamberlain, 2000). Indeed, restriction of glyphosate delivery to the plant cell cytoplasm (and chloroplast) by any means is, in itself, a resistance mechanism (Shaner, 2009; Ge et al., 2013). Elucidation of key factors governing delivery to the intracellular milieu of plant source leaves is critical for developing a complete understanding of the mechanism(s) of resistance to glyphosate.Glyphosate’s phosphono group offers the opportunity to employ in vivo ³¹P-NMR spectroscopy to track glyphosate movement and metabolism, additionally including monitoring of cellular pH, and gradients therein, and ATP levels, both indicators of tissue viability (Roberts, 1984). This laboratory has extended the ³¹P-NMR approach initially used by Gout et al. (1992) with suspension-cultured sycamore (Acer pseudoplatanus) cells. The initial findings, that source and sink leaf tissue from GR horseweed rapidly and avidly sequestered glyphosate within the vacuole compartment and that leaf tissue from glyphosate-sensitive (GS) horseweed did not, led to the hypothesis that vacuole sequestration was a key, perhaps the dominant, component of the resistance mechanism (Ge et al., 2010). It was then shown that GR horseweed acclimated and maintained at cold temperature (approximately 10°C–12°C) did not rapidly and avidly sequester glyphosate within the vacuole. Importantly, under such conditions, GR horseweed succumbed to the toxic effects of glyphosate. In short, by preventing glyphosate sequestration, GR horseweed became glyphosate sensitive, a laboratory finding confirmed in the field (Ge et al., 2011).The proposition that, by limiting the herbicide available for translocation, glyphosate vacuole sequestration could serve as an important if not dominant resistance mechanism was further strengthened by experiments that showed vacuolar glyphosate sequestration correlated with glyphosate resistance in ryegrass (Lolium spp.) from Australia, South America, and Europe (Ge et al., 2012). However, ³¹P-NMR studies of other weeds revealed that in some species, for example, Palmer amaranth, waterhemp (Amaranthus tuberculatus), and johnsongrass (Sorghum halepense), resistance correlated strongly with a lack of glyphosate uptake into the plant cell, a frontline resistance mechanism (Ge et al., 2013).Throughout these previous ³¹P-NMR studies, the finding that plants could regulate the compartmental access of glyphosate led us to speculate that the apoplast, tonoplast, and perhaps chloroplast possessed glyphosate-active transporters whose up-regulation or down-regulation and/or expression would confer resistance (Ge et al., 2010, 2011, 2012, 2013). This hypothesis motivated additional in vivo ³¹P-NMR experiments to further describe the determinants of glyphosate delivery in horseweed leaf tissue. Specifically, experiments with GR horseweed were designed with the goal of probing the transporter hypothesis.Findings from these experiments are reported herein and are consistent with the existence of a tonoplast transporter that is responsible for glyphosate resistance via vacuole sequestration. As described here, vacuole sequestration requires ATP, is active for multiple substrates, and shows substrate competition. Furthermore, glyphosate entry into the cell can be markedly inhibited by vanadate pretreatment. These characteristics are similar to those of ATP-binding cassette transporters in plants (Hetherington et al., 1998; Rea, 2007; Verrier et al., 2008; Prosecka et al., 2009; Conte and Lloyd, 2011) and mammalian cells (van de Ven et al., 2009; Ernst et al., 2010).