NanoScience Technology Center, University of Central Florida, Orlando, FL, 32826, USA.
Department of Chemistry, University of Central Florida, Orlando, FL, 32826, USA.
Planta. 2021 Feb 5;253(2):62. doi: 10.1007/s00425-020-03559-z.
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. 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.
基于荧光团标记抗生素缀合物的激发态寿命与光谱宽的植物自发荧光分离，使得通过荧光寿命成像对系统货物（如抗生素）的易位进行体内追踪成为可能。由于田间实验的结果不一致，以及对其系统运动缺乏研究，柑橘作物中抗生素治疗的效果尚不确定。到目前为止，由于传统荧光显微镜无法追踪治疗方法，因为治疗方法荧光特征很少，并且由于植物自发荧光而导致信号卷积。在这项研究中，我们使用硫酸链霉素，一种市售的抗生素，并将其与一种改良的三（联吡啶）钌（II）氯化物缀合，该染料的激发态寿命比其他常用的有机荧光探针大得多。由此产生的是一种荧光寿命成像（FLIM）可追踪的抗生素缀合物，通过酰胺键共价连接，与植物自发荧光具有独特的可区分性。荧光抗生素缀合物的特性表明其激发态寿命没有降低，并且不存在任何合成成分中发现的独特的 IR 峰。随后在柑橘组织中使用 FLIM 进行跟踪，通过在木质部和韧皮部中的组织定位来识别在柑橘植物脉管系统中的运动。结果表明，在孵育 48 小时后，缀合物在木质部和韧皮部中都向上进行系统运动。然而，在 168 小时后，缀合物未能向下移动到植物的根系。从机制上讲，木质部很可能在缀合物向上转移中起重要作用；然而，由于生长变化，韧皮部导致的流可能是一个促成因素。这个概念验证为后续关于柑橘中抗生素定位和运动的研究奠定了基础。
