Novel bioassay for the discovery of inhibitors of the 2-C-methyl-D-erythritol 4-phosphate (MEP) and terpenoid pathways leading to carotenoid biosynthesis.

The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.

Roots of the invasive species Carduus nutans L. and C. acanthoides L. produce large amounts of aplotaxene, a possible allelochemical.

The invasive thistle Carduus nutans has been reported to be allelopathic, yet no allelochemicals have been identified from the species. In a search for allelochemicals from C. nutans and the closely related invasive species C. acanthoides, bioassay-guided fractionation of roots and leaves of each species were conducted. Only dichloromethane extracts of the roots of both species contained a phytotoxin (aplotaxene, (Z,Z,Z)-heptadeca-1,8,11,14-tetraene) with sufficient total activity to potentially act as an allelochemical. Aplotaxene made up 0.44 % of the weight of greenhouse-grown C. acanthoides roots (ca. 20 mM in the plant) and was not found in leaves of either species. It inhibited growth of lettuce 50 % (I 50) in soil at a concentration of ca. 0.5 mg g(-1) of dry soil (ca. 6.5 mM in soil moisture). These values gave a total activity in soil value (molar concentration in the plant divided by the molarity required for 50 % growth inhibition in soil = 3.08) similar to those of some established allelochemicals. The aplotaxene I 50 for duckweed (Lemna paucicostata) in nutrient solution was less than 0.333 mM, and the compound caused cellular leakage of cucumber cotyledon discs in darkness and light at similar concentrations. Soil in which C. acanthoides had grown contained aplotaxene at a lower concentration than necessary for biological activity in our short-term soil bioassays, but these levels might have activity over longer periods of time and might be an underestimate of concentrations in undisturbed and/or rhizosphere soil.