Development of a rapid biolistic assay to determine changes in relative levelsof intracellular calcium in leaves following tetracycline uptake by pinto beanplants

摘要

Tetracycline antibiotics, such as chlortetracycline (CTC) and tetracycline (TC), are introduced intoagricultural lands through the application of manure as fertilizer. These compounds are phytotoxic tocertain crop plants, including pinto beans (Phaseolus vulgaris), the species used for this investigation.While the mechanism of this toxicity is not yet understood, CTC is known to be a calcium chelator. Wedescribe here a novel method to show that CTC is taken up by pinto bean plants and chelates calcium inleaves. Cameleon fusion proteins can provide qualitative and quantitative imaging of intracellularcalcium levels, but current methodology requires stable transformation. Many plant species, includingpinto beans, are not yet transformable using standard Agrobacterium-based protocols. To determinethe role of calcium chelation in this plant, a rapid, biolistic method was developed to transiently expressthe cameleon protein. This method can easily be adapted to other plant systems. Our findings provideevidence that chelation of intracellular calcium by CTC is related to phytotoxic effects caused by thisantibiotic in pinto beans. Root uptake of CTC and TC by pinto beans and their translocation to leaveswere further verified by fluorescence spectroscopy and liquid chromatography/mass spectrometry,confirming results of the biolistic method that showed calcium chelation by tetracyclines in leaves.