Large‐scale screening of Arabidopsis circadian clock mutants by a high‐throughput real‐time bioluminescence monitoring system

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Abstract
Using a high-throughput real-time bioluminescence monitoring system, we screened large numbers of Arabidopsis thaliana mutants for extensively altered circadian rhythms. We constructed reporter genes by fusing a promoter of an Arabidopsis flowering-time gene - either GIGANTEA (GI) or FLOWERING LOCUS T (FT) - to a modified firefly luciferase gene (LUC(+)), and we transferred the fusion gene (P(GI)::LUC(+) or P(FT)::LUC(+)) into the Arabidopsis genome. After mutagenesis with ethyl methanesulfonate, 50 000 M(2) seedlings carrying the P(GI)::LUC(+) and 50 000 carrying P(FT)::LUC(+) were screened their bioluminescence rhythms. We isolated six arrhythmic (AR) mutants and 29 other mutants that showed more than 3 h difference in the period length or phase of rhythms compared with the wild-type strains. The shortest period length was 16 h, the longest 27 h. Five of the six AR mutants carrying P(GI)::LUC(+) showed arrhythmia in bioluminescence rhythms in both constant light and constant dark. These five AR mutants also showed arrhythmia in leaf movement rhythms in constant light. Genetic analysis revealed that each of the five AR mutants carried a recessive mutation in a nuclear gene and the mutations belonged to three complementation groups, and at least one of which was mapped on a novel locus. Our results suggest that the three loci identified here may contain central clock or clock-related genes, at least one of which may be a novel.