Cytokinins are one kind of phytohormones essential for plant growth, development and stress responses. In the past half century, significant progresses have been made in the studies of cytokinin signal transduction and metobolic pathways, but the mechanism of cytokinin translocation is poorly understood. Arabidopsis (Arabidopsis thaliana) response regulator 5 (ARR5) is a type-A response factor in cytokinin signaling which is induced by cytokinins and has been used as a reporter gene for the endogenous cytokinins in Arabidopsis. Here, we report a fluorescence-based high-throughput method to screen cytokinin translocation mutants using an ethyl methyl sulfone (EMS) mutagenesis library generated with ARR5::eGFP transgenic plants. The seedlings with enhanced green fluorescent protein (GFP) signal in roots were screened in a luminescence imaging system (LIS) in large scale to obtain mutants with over-accumulated cytokinins in roots. The selected mutants were confirmed under a fluorescence microscopy and then performed phenotypic analysis. In this way, we obtained twelve mutants with elevated GFP signal in the roots and further found three of them displayed reduced GFP signal in the aerial tissues. Two of the mutants were characterized and proved to be the atabcg14 allelic mutants which are defective in the long-distance translocation of root-synthesized cytokinins. We provide a strategy for screening mutants defective in cytokinin translocation, distribution or signaling. The strategy can be adapted to establish a system for screening mutants defective in other hormone transporters or signaling components using a fluorescence reporter.

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基于荧光的细胞分裂素易位突变体高通量筛选方法

细胞分裂素是植物生长、发育和应激反应所必需的一种植物激素。在过去的半个世纪里，细胞分裂素信号转导和代谢途径的研究取得了重大进展，但细胞分裂素易位的机制却知之甚少。拟南芥 (Arabidopsis thaliana) 反应调节因子 5 (ARR5) 是细胞分裂素信号传导中的 A 型反应因子，由细胞分裂素诱导，已被用作拟南芥内源性细胞分裂素的报告基因。在这里，我们报告了一种基于荧光的高通量方法，使用 ARR5::eGFP 转基因植物生成的乙基甲基砜 (EMS) 诱变库筛选细胞分裂素易位突变体。在发光成像系统（LIS）中大规模筛选根部具有增强的绿色荧光蛋白（GFP）信号的幼苗，以获得根部具有过度积累的细胞分裂素的突变体。在荧光显微镜下确认选择的突变体，然后进行表型分析。通过这种方式，我们获得了 12 个根中 GFP 信号升高的突变体，进一步发现其中 3 个在地上组织中显示出 GFP 信号降低。其中两个突变体被鉴定并证明是atabcg14等位基因突变体，它们在根合成的细胞分裂素的长距离易位中存在缺陷。我们提供了一种筛选细胞分裂素易位、分布或信号传导缺陷突变体的策略。