Microarray Analysis of Diurnal and Circadian-Regulated Genes in Arabidopsis

Abstract

Plants respond to day/night cycling in a number of physiological ways. At the mRNA level, the expression of some genes changes during the 24-hr period. To identify novel genes regulated in this way, we used microarrays containing 11,521 Arabidopsis expressed sequence tags, representing an estimated 7800 unique genes, to determine gene expression levels at 6-hr intervals throughout the day. Eleven percent of the genes, encompassing genes expressed at both high and low levels, showed a diurnal expression pattern. Approximately 2% cycled with a circadian rhythm. By clustering microarray data from 47 additional nonrelated experiments, we identified groups of genes regulated only by the circadian clock. These groups contained the already characterized clock-associated genes LHY , CCA1 , and GI , suggesting that other key circadian clock genes might be found within these clusters. INTRODUCTIONPlants have adapted their growth and development to use the diurnal cycling of light and dark. This is manifested at both the physiological level, with leaf movement, growth, and stomatal opening, and the molecular level, with expression of some genes occurring only at certain times of the day. The day/night cycling of gene expression is called a diurnal rhythm and is achieved primarily by two mechanisms: first, by light, and second, by a free-running internal circadian clock. Circadian clocks have been well characterized in animals, fungi, and bacteria, and in all cases they have a central oscillator that measures time with a molecular feedback loop that cycles over a 24-hr period (Dunlap, 1999). Although a growing number of genes either regulated by the clock or affecting clock function have been identified in plants, a full picture has yet to emerge.The ability of plants to respond to light is achieved through photoreceptors. In Arabidopsis, two classes of photoreceptors are known: the red/far-red receptors, phytochrome A to E (Sharrock and Quail, 1989;Clack et al., 1994), and the blue light receptors, CRY1 (Ahmad and Cashmore, 1993), CRY2 (Guo et al., 1998), and NPH1 (Liscum and Briggs, 1995). Using these photoreceptors, a plant can detect a range of light intensities and wavelengths, with which it senses not only whether light is present but also from which direction the light is coming and whether there is competing vegetation (reviewed in Ballare, 1999). The best characterized of the photoreceptors are the phytochromes, for which the events that convert the light signal into transcriptional regulation have been described. Phytochrome is transported into the nucleus in a light-dependent manner (Sakamoto and Nagatani, 1996;Kircher et al., 1999). In the nucleus, it interacts with a basic helix-loop-helix transcription factor, PIF3 (Ni et al., 1998(Ni et al., , 1999, which has been shown to bind to the G box element found in the promoters of many light-activated genes (Giuliano et al., 1988;Martinez-Garcia et al., 2000). This chain of events allows the plants to respond to light after germination, by stopping hypocotyl elongat...

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