1 Scientific RepoRts | 6:27333 | DOI: 10.1038/srep27333 www.nature.com/scientificreports Chalcone-based selective Inhibitors of a C 4 plant Key enzyme as Novel potential Herbicides G. t. t. Nguyen 1 , G. erlenkamp 2 , o. Jäck 3 , A. Küberl 4 , M. Bott 4 , F. Fiorani 3 , H. Gohlke 2 & G. Groth 1 Weeds are a challenge for global food production due to their rapidly evolving resistance against herbicides. We have identifed chalcones as selective inhibitors of phosphoenolpyruvate carboxylase (PEPC), a key enzyme for carbon fxation and biomass increase in the C 4 photosynthetic pathway of many of the world’s most damaging weeds. In contrast, many of the most important crop plants use C 3 photosynthesis. Here, we show that 2′,3′,4′,3,4-Pentahydroxychalcone (IC 50 = 600 nM) and 2′,3′,4′-Trihydroxychalcone (IC 50 = 4.2 μM) are potent inhibitors of C 4 PEPC but do not afect C 3 pepC at a same concentration range (selectivity factor: 15–45). Binding and modeling studies indicate that the active compounds bind at the same site as malate/aspartate, the natural feedback inhibitors of the C 4 pathway. At the whole plant level, both substances showed pronounced growth-inhibitory efects on the C 4 weed Amaranthus retrofexus, while there were no measurable efects on oilseed rape, a C 3 plant. Growth of selected soil bacteria was not afected by these substances. Our chalcone compounds are the most potent and selective C 4 PEPC inhibitors known to date. They ofer a novel approach to combat C 4 weeds based on a hitherto unexplored mode of allosteric inhibition of a C 4 plant key enzyme. Worldwide, weeds cause more yield loss and add more to farmers’ production costs than any other agricultural pest. Tis weed challenge on global food production has become severe due to rapidly evolving resistance of many weed species, resulting in resistance against herbicides addressing 22 of the 25 molecular targets known to date for weed control. Many of the worst weeds use C 4 photosynthesis, whereas the majority of crop plant species use the classical C 3 photosynthetic pathway. Hence, enzymes of the C 4 pathway provide an excellent target to combat these weeds. In C 4 photosynthesis, inorganic carbon is initially fxed by the enzyme phosphoenolpyruvate (PEP) carboxylase (PEPC), yielding the four-carbon molecule oxaloacetate 1 . Oxaloacetate is then reduced to malate or transaminated to aspartate in the decarboxylation reaction of the C 4 pathway 2 . Tere is a major diference in the sensitivity of PEPC from C 3 and C 4 plants towards feedback inhibition by C 4 carboxylates from the C 4 pathway 3 . Previously, we selected the genus Flaveria as a model to study the feedback inhibitor tolerance of PEPC of C 3 and C 4 plants 4 because this genus contains various C 3 , C 3 -C 4 intermediate, and C 4 species 5 . A single residue in the dicarboxylate feedback inhibitor binding site was shown to control the diferent malate tolerance of C 3 and C 4 plants 4 : Arginine-884 of F. pringlei (C 3 plant) PEPC assists the feedback inhibitor binding, whereas glycine at the same position of F. trinervia (C 4 plant) PEPC forms no interaction with the inhibitor 4 . Arginine-884 is conserved in all typical C 3 crop plants. In most C 4 weeds, glycine, serine, or glutamine are found in this position 6 . Hence, the molecular diference in the feedback inhibitor binding site of PEPC in C 3 and C 4 plants should allow developing selective herbicides for weed control. We showed that catechins and quinoxalines are selective C 4 PEPC inhibitors with IC 50 values in the range of 100 μM 7 . However, small molecule compounds with enhanced inhibitory efects and selectivity for C 4 PEPC are required to advance further development of C 4 selective herbicides. In this study, based on their chemical and structural similarity with the previously introduced C 4 -selective catechine inhibitors 7 , we identify members of the chalcone family from chemical libraries as suitable 1 Biochemical Plant Physiology, Heinrich Heine University Düsseldorf and Bioeconomy Science center (BioSc), Universitätsstr.1, 40225 Düsseldorf, Germany. 2 Pharmaceutical and Medicinal chemistry, Heinrich Heine University Düsseldorf and Bioeconomy Science Center (BioSC), Universitätsstr.1, 40225 Düsseldorf, Germany. 3 institute of Bio- and Geosciences, IBG-2: Plant Sciences, Forschungszentrum Jülich and Bioeconomy Science Center (BioSC), Wilhelm-Johnen-Straße, 52425 Jülich, Germany. 4 Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich and Bioeconomy Science Center (BioSC), Wilhelm-Johnen-Straße, 52425 Jülich, Germany. Correspondence and requests for materials should be addressed to G.G. (email: Georg.Groth@hhu.de) received: 03 February 2016 accepted: 18 May 2016 Published: 06 June 2016 opeN