Imaging of glyphosate uptake and identification of early microscopic markers in leaves of C3 and C4 glyphosate-resistant and -susceptible species.

Details of glyphosate uptake are not fully elucidated, and although this herbicide promotes important alterations in the plant phenotype few hours after its application (early responses), a detailed description of the presumable changes in plant anatomy is still poorly assessed by now. Due to glyphosate effects over leaf permeability, the use of an inert fluorescent tracer may allow the observation of the uptake event in situ. In addition, microscopic markers might put a light on the recognition of glyphosate-resistant (GR) and -susceptible (GS) species or varieties, which may vary in C3 and C4 species due to their putative distinct leaf anatomy. Here we aimed (i) to provide a new technique to track the route of glyphosate formulation towards leaf tissues using a fluorescent tracer, and (ii) to describe the early specific microscopic alterations in GR and GS -C3 or -C4 caused by the glyphosate formulation. Roundup Transorb was applied in seedlings cultivated in a greenhouse and response alterations in leaf anatomy were described. Lucifer Yellow CH (LYCH) was applied over the same region where glyphosate formulation was previously applied to track the alterations in leaf permeability caused by this herbicide. LYCH successfully tracked the glyphosate formulation uptake, reaching the vascular bundles of GS species, and becoming retained in leaf tissues of GR species. All species exhibited a decrease in chlorophyll content at the site of glyphosate application regardless of their photosynthetic metabolism or susceptibility. GS species showed alterations in chloroplast morphology and activity of non-enzymatic antioxidants (carotenoids and flavonoids), in addition to symptoms indicating a process of accelerated cell senescence. A specific type of cell necrosis (hypersensitive response) was observed in GR-C4 species as a way to prevent the translocation of this herbicide, while GR-C3 species accumulated phenolic compounds inside the vacuole, probably sequestrating and inactivating the glyphosate action. This study provides a reliable tool to track glyphosate formulation uptake in situ and is the first attempt to the identification of early specific microscopic markers caused by glyphosate formulation.