Identification of MDH as a herbicidal protein target of botanical D-pinitol in Amaranthus retroflexus L.

Natural products constitute a significant reservoir for novel pesticide development. D -pinitol ((1 R,2S,3r,4 R,5S,6r)-6-methoxycyclohexane-1,2,3,4,5-pentol), a compound exhibiting robust herbicidal activity, has been isolated from Sapindus mukorossi leaf extracts. Nevertheless, its precise molecular target remains unidentified. This study elucidates the targets of D -pinitol in A. retroflexus through an integration of Drug Affinity Responsive Target Stability (DARTS), affinity chromatography, and transcriptomics. DARTS and affinity chromatography revealed 18 and 25 principal binding proteins, respectively. Integrating these findings with transcriptomics results identified potential target proteins: Serine hydroxy methyltransferase (SHMT), Ketol-acid reductor-isomerase (KARI), Isocitrate dehydrogenase (IDH), Malate dehydrogenase (MDH), Acetyl-CoA carboxylase (ACC), Alpha tubulin (α-tubulin), and Dihydrolipoamide dehydrogenase (DLD). Molecular docking and fluorescence quenching analysis further pinpointed MDH as the probable molecular target. D -pinitol demonstrated specific binding affinity to MDH, with dissociation equilibrium constants (K D ) of 5.17 μM and 1.34 μM determined by surface plasmon resonance (SPR) and microscale thermophoresis (MST), respectively. Site-directed mutagenesis combined with fluorescence quenching analysis indicated that residues GLN-67 and GLY-110 are crucial for D -pinitol binding to MDH. This research identifies MDH as the initial molecular target of D -pinitol, thereby providing a foundation for designing and synthesizing novel green herbicides. • The target protein of D -pinitol was identified as MDH. • The binding ability of D -pinitol and MDH was verified. • The key amino acid binding sites for D -pinitol and MDH were identified. • This study provides a new target for the study of green herbicide.