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The identification of novel herbicides is of crucial importance to modern agriculture. We developed an efficient  in vivo  assay based on oxygen evolution measurements using suspensions of chlorenchyma cells isolated from the single-cell C 4  plant  Bienertia sinuspersici  to identify and characterize inhibitors of C 4  photosynthesis. This novel approach fills the gap between conventional  in vitro  assays for inhibitors targeting C 4  key enzymes and  in vivo  experiments on whole plants. The assay addresses inhibition of the target enzymes in a plant context thereby taking care of any reduced target inhibition due to metabolization or inadequate uptake of small molecule inhibitors across plant cell walls and membranes. Known small molecule inhibitors targeting C 4  photosynthesis were used to validate the approach. To this end, we tested pyruvate phosphate dikinase inhibitor bisindolylmaleimide IV and phosphoenolpyruvate carboxylase inhibitor okanin. Both inhibitors show inhibition of plant photosynthesis at half-maximal inhibitory concentrations in the sub-mM range and confirm their potential to act as a new class of C 4  selective inhibitors.

frontiers in plant science

Efficient In Vivo Screening Method for the Identification of C4 Photosynthesis Inhibitors Based on Cell Suspensions of the Single-Cell C4 Plant Bienertia sinuspersici