Engineering glyphosate tolerance in sugarcane through overexpression of a mutated version of its native EPSPS gene

Sugarcane is a globally important crop, widely used in both the food industry and biofuel production. Weed infestations can significantly reduce its productivity, and to address this challenge, herbicides such as glyphosate are commonly applied. Glyphosate acts as a structural analog of phosphoenolpyruvate (PEP), inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), thereby disrupting the shikimate pathway and impairing the biosynthesis of essential aromatic amino acids required for plant growth and development. However, the limited tolerance of sugarcane to glyphosate restricts herbicide application to doses that are often insufficient for effective weed control. Previous studies have shown that amino acid substitutions in the EPSPS enzyme, particularly the T102I/P106A (TIPA) mutations, can confer glyphosate resistance in plants. In this study, we evaluated whether a specific mutation in the endogenous ScEPSPS gene could enhance glyphosate tolerance in sugarcane without compromising morphological, physiological, and biometric parameters evaluated under greenhouse conditions. The mutated gene was constitutively overexpressed in transgenic sugarcane plants, which survived glyphosate concentrations as high as 5% (v/v of the commercial formulation, 106.5 mM of glyphosate acid equivalent). Molecular analyses revealed approximately 25- to 80-fold overexpression of the transgene in resistant lines compared with non-transgenic controls. High-performance liquid chromatography (HPLC) quantification showed elevated levels of phenylalanine, tyrosine, and tryptophan 14 days after herbicide application in plants overexpressing the mutated ScEPSPS. Although the mutation affected certain histological and physiological parameters, biometric assessments indicated that the overall phenotype remained largely unchanged. The successful overexpression of the mutated endogenous ScEPSPS gene represents a promising biotechnological strategy to enhance glyphosate tolerance in sugarcane without compromising plant growth and development under greenhouse conditions. The overexpression of the ScEPSPS gene bearing the T102I/P106A (TIPA) mutation confers resistance to concentrations of up to 5% glyphosate in sugarcane plants, without compromising their agronomic functions.