Functional Conservation of Clock-Related Genes in Flowering Plants: Overexpression and RNA Interference Analyses of the Circadian Rhythm in the Monocotyledon Lemna gibba

Circadian rhythms are found in organisms from cyanobacteria to plants and animals. In flowering plants, the circadian clock is involved in the regulation of various physiological phenomena, including growth, leaf movement, stomata opening, and floral transitions. Molecular mechanisms underlying the circadian clock have been identified using Arabidopsis (Arabidopsis thaliana); the functions and genetic networks of a number of clock-related genes, including CIRCADIAN CLOCK ASSOCIATED1, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1, GIGANTEA (GI), and EARLY FLOWERING3 (ELF3), have been analyzed. The degree to which clock systems are conserved among flowering plants, however, is still unclear. We previously isolated homologs for Arabidopsis clock-related genes from monocotyledon Lemna plants. Here, we report the physiological roles of these Lemna gibba genes (LgLHYH1, LgLHYH2, LgGIH1, and LgELF3H1) in the circadian system. We studied the effects of overexpression and RNA interference (RNAi) of these genes on the rhythmic expression of morning- and evening-specific reporters. Overexpression of each gene disrupted the rhythmicity of either or both reporters, suggesting that these four homologs can be involved in the circadian system. RNAi of each of the genes except LgLHYH2 affected the bioluminescence rhythms of both reporters. These results indicated that these homologs are involved in the circadian system of Lemna plants and that the structure of the circadian clock is likely to be conserved between monocotyledons and dicotyledons. Interestingly, RNAi of LgGIH1 almost completely abolished the circadian rhythm; because this effect appeared to be much stronger than the phenotype observed in an Arabidopsis gi loss-of-function mutant, the precise role of each clock gene may have diverged in the clock systems of Lemna and Arabidopsis.