Abstract
BackgroundCytokinins 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.ResultsThe 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.ConclusionsWe 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.
Highlights
Cytokinins are one kind of phytohormones essential for plant growth, development and stress responses
Design of an ARR5::eGFP reporter‐based approach for screening cytokinin translocation mutants To further understand the mechanism of cytokinin shootward translocation, we designed a forward genetic approach for screening cytokinin translocation mutants
A flow chart was presented to screen ethyl methane sulfonate (EMS)-based mutants defective in the shootward translocation of cytokinins (Fig. 1). tZ-type cytokinins are synthesized in the root tip of ARR5::eGFP Col-4 and transported to the shoot through xylem for plant growth and development [11, 26]
Summary
Cytokinins are one kind of phytohormones essential for plant growth, development and stress responses. Cytokinins regulate multiple life processes of plants such as leaf senescence and stress responses [3,4,5]. 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]. The metabolite analysis, transcriptome and grafting experiments showed that the tZ and DHZ are mainly produced in the root and moved acropetally while the iP-type cytokinins in the shoot can be moved basipetally or systemically [11,12,13,14]
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