However, ensuring homogeneous illumination of all leaf cuttings for all those repetitions and across different studies still remains challenging in this set-up

However, ensuring homogeneous illumination of all leaf cuttings for all those repetitions and across different studies still remains challenging in this set-up. the approach. To this end, we tested pyruvate phosphate dikinase inhibitor bisindolylmaleimide IV and phosphoenolpyruvate carboxylase inhibitor okanin. Both inhibitors show inhibition of herb photosynthesis at half-maximal inhibitory concentrations in the sub-mM range and Povidone iodine confirm their potential to act as a new class of C4 selective inhibitors. or utilize C4 photosynthesis. With Povidone iodine the rise of known resistances of weeds to commercially available herbicides over the past decades the effects of resistant weeds on modern agriculture are becoming an increasingly pressing issue (Heap, 2014; Busi et al., 2018; Heap, 2019). In addition to herbicide resistances, the predicted increase in average temperatures caused by global warming and rising levels of atmospheric CO2 are thought to shift the odds in favor of C4 plants which will eventually render C4 weeds even more competitive in the context of C3 crops (Fernando et al., 2016; Carboni et al., 2017; Waryszak et al., 2018). To combat this development, new herbicides that are specifically designed to target C4 weeds are needed. The identification and characterization of novel inhibitors interfering with mandatory biochemical pathways in plants are key requirements for the development of new herbicides. This process usually includes assays which assess the efficiency and efficacy toward a specific target enzyme followed by extensive Gpc4 studies using whole plants. However, compounds that prove to be effective may not be effective at all on whole plants due to reduced bioavailability which might be caused by several issues such as slow uptake into the herb tissue, solubility problems or detoxification of the active compound within the cells (Shimabukuro, 1985). Furthermore, studies with whole plants are cumbersome particularly in terms of set-up and repetitions, human resources, and technical facilities required for a controlled environment. Consequently, an alternative method for the preliminary validation of biological effectiveness of compounds is needed for pre-screening purposes. In recent years, a number of inhibitory compounds targeting C4 photosynthesis have been identified (Haines et al., 2005; Nguyen et al., 2016; Dick et al., 2017; Minges and Groth, 2017). These compounds inhibit PPDK or PEPC activity, which are key enzymes in Kranz C4 and SCC4 pathways. Effects of C4 inhibitors have been previously studied using purified enzymes in spectrophotometric assays (Doyle et al., 2005; Motti et al., 2007; Nguyen et al., 2016; Minges and Groth, 2017). While experiments are usually fast and convenient to perform and scale-up well (Feng et al., 2005; Bailey et al., 2018), they represent a dramatic simplification of the larger biological context in which the studied biochemical reaction takes place. Here, herb related metabolization or uptake limitations of the compound due to the herb cuticula, cell walls or cell membrane are largely ignored. These restrictions are avoided when leaf cuttings or whole plants are used and compounds are tested at conditions (Haines et al., 2005; Motti et al., 2007). Compared to whole herb toxicity assays, oxygen evolution measurements on leaf cuttings are relatively easy and straightforward to perform using Clark-type electrodes with buffer-filled reaction chambers (Clark, 1956; Delieu and Walker, 1972). In this set-up the cuticular barrier is usually bypassed as water-soluble compounds are able to freely diffuse Povidone iodine into the cells and within the symplast by the plasmodesmata uncovered on the cut surfaces. Nevertheless, herb related metabolization of the compound may be still observable in these studies as most of the herb tissue is still largely intact. However, ensuring homogeneous illumination of all leaf cuttings for all those repetitions and across different studies.