Abstract
Main conclusion
Excited state lifetime-based separation of fluorophore-tagged antibiotic conjugate emission from the spectrally broad plant autofluorescence enables in planta tracking of the translocation of systemic cargo such as antibiotics via fluorescence lifetime imaging.
Abstract
The efficacy of antibiotic treatments in citrus crops is uncertain due to mixed results from in-field experiments and a lack of study on their systemic movement. As of yet there has been an inability to track treatments using traditional fluorescence microscopy due to treatments having little fluorescence characteristics, and signal convolution due to plant autofluorescence. In this study, we used streptomycin sulfate, a commercially available antibiotic, and conjugated it to a modified tris(bipyridine) ruthenium (II) chloride, a dye with an excited state lifetime magnitudes higher than other commonly used organic fluorescent probes. The resultant is a fluorescence lifetime imaging (FLIM) trackable antibiotic conjugate, covalently attached via an amide linkage that is uniquely distinguishable from plant autofluorescence. Characterization of the fluorescent antibiotic conjugate showed no mitigation of excited state lifetime, and a distinct IR peak not found in any synthetic components. Subsequent tracking using FLIM in citrus tissue was achieved, with identification of movement through citrus plant vasculature via tissue localization in xylem and phloem. Results indicated upwards systemic movement of the conjugate in both xylem and phloem after 48 h of incubation. However, the conjugate failed to move down towards the root system of the plant by 168 h. Mechanistically, it is likely that xylem contributes heavily in the translocation of the conjugate upwards; however, phloem led flow due to growth changes could act as a contributor. This proof-of-concept sets groundwork for subsequent studies regarding antibiotic localization and movement in citrus.
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Acknowledgements
The authors would like to acknowledge Dr. Evan Johnson and Tony Macintosh for providing citrus saplings, Dr. Maria Campos for her help with utilizing the growth chamber, Dr. Titel Jurca for use of glassware for synthesis of the conjugate, and Dr. Matthew Rex for his assistance in running HPLC. This work was supported by the National Institute of Food and Agriculture [Grant no. 2015-70016-23010/project accession no. 1005557] from the USDA National Institute of Food and Agriculture.
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Communicated by Anastasios Melis.
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Miller, G.S., Parente, R.M., Santra, S. et al. Tracking of fluorescent antibiotic conjugate in planta utilizing fluorescence lifetime imaging. Planta 253, 62 (2021). https://doi.org/10.1007/s00425-020-03559-z
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DOI: https://doi.org/10.1007/s00425-020-03559-z