The herbicide 2,4-D exhibits an auxinic activity and therefore can be used as a synthetic and traceable analog to study auxin-related responses. Here we identified that not only exogenous 2,4-D but also its amide-linked metabolite 2,4-D-Glu displayed an inhibitory effect on plant growth via the TIR1/AFB auxin-mediated signaling pathway. To further investigate 2,4-D metabolite conversion, identity and activity, we have developed a novel purification procedure based on the combination of ion exchange and immuno-specific sorbents combined with a sensitive liquid chromatography-mass spectrometry method. In 2,4-D treated samples, 2,4-D-Glu and 2,4-D-Asp were detected at 100-fold lower concentrations compared to 2,4-D levels, showing that 2,4-D can be metabolized in the plant. Moreover, 2,4-D-Asp and 2,4-D-Glu were identified as reversible forms of 2,4-D homeostasis that can be converted to free 2,4-D. This work paves the way to new studies of auxin action in plant development.

Department of Applied Genetics and Cell Biology University of Natural Resources and Life Sciences Vienna Austria

Department of Chemical Biology and Genetics Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science of Palacký University Olomouc Czech Republic

Department of Forest Genetics and Plant Physiology Umeå Plant Science Centre Swedish University of Agricultural Sciences Umeå Sweden

Department of Virology Veterinary Research Institute Brno Czech Republic

Laboratory of Growth Regulators Centre of the Region Haná for Biotechnological and Agricultural Research Institute of Experimental Botany CAS and Faculty of Science of Palacký University Olomouc Czech Republic

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