A fluorescence-based high-throughput screening method for cytokinin translocation mutants
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Plant Methods Open Access
METHODOLOGY
A fluorescence‑based high‑throughput screening method for cytokinin translocation mutants Mengyuan Zhang1,2, Bingli Ding1,2, Jiangzhe Zhao1,2, Penghong Zhang1,2, Yujia Li1,2, Guodong Yang3 and Kewei Zhang1,2*
Abstract Background: 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. Results: 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. Conclusions: 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. Keywords: Cytokinin, Translocation, Arabidopsis response regulator 5, EMS mutagenesis, Fluorescence reporter Background Cytokinins are one kind of phytohormones defined by their functions of promoting plant cell division and differentiation [1, 2]. Cytokinins also regulate multiple life processes of plants such as leaf senescence and stress responses [3–5]. They were chemically identified as *Correspondence: [email protected] 1 Institute of Plant Genetics and Developmental Biology, College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China Full list of author information is available at the end of the article
adenine derivatives and mainly classified as trans-zeatin (tZ), N6-(Δ2-isopentenyl) adenine (iP), cis-zeatin (cZ) and dihydrozeatin (DHZ) types by their multiple side-chains at N6 position of the adenine backbone [4]. The biosynthesis of cytokinins in Arabidopsis (Arabidopsis thaliana) is well understood and the adenosine phosphate-isopentenyltransferases (IPTs) are responsible for the initial step to produce iP nucleotides in the pathway [6, 7]. T
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