Multiple mechanism confers natural tolerance of three lilyturf species to glyphosate

MAIN CONCLUSION : A combination of unique EPSPS structure and increased gene copy number and expression contribute to natural glyphosate tolerance in three lilyturf species. A few plants are naturally tolerant to glyphosate, the most widely used non-selective herbicide worldwide. Here, the basis for natural tolerance to glyphosate in three lilyturf species, Ophiopogon japonicus (OJ), Liriope spicata (LS), and Liriope platyphylla (LP), is characterized. These species tolerate glyphosate at about five times the commercially recommended field dose. They share three unique amino acids in their 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) that affect glyphosate binding. These correspond to Asp71Met, Ala112Ile, and Val201Met amino acid variations compared to 231 other published plant EPSPS amino acid sequences. There was also a common deletion at 91 of a highly conserved glutamic acid. Glyphosate-treated lilyturf plants accumulated little shikimic acid but had significantly higher levels of EPSPS mRNA than initially expressed in the control. The IC₅₀ of LsEPSPS was 14.0 µM compared to the 5.1 µM of Arabidopsis thaliana. The higher K ₘ and K ᵢ values of LsEPSPS kinetics showed that LsEPSPS had lower substrate binding affinity to glyphosate. Overexpression of LsEPSPS in the recombinant E. coli BL21 (DE3) strain enhanced its tolerance to glyphosate. Both OJ and LS had two copies of the EPSPS gene, while LP had three copies. Therefore, a combination of unique EPSPS structure and increased gene copy number and expression contribute to natural glyphosate tolerance in the three lilyturf species.