TOC1 was the first plant gene that, when mutated, yielded a circadian phenotype. It codes for the transcription factor TOC1, which affects the period of plants’ circadian rhythms: built-in, malleable oscillations that repeat every 24 hours. The gene codes for a transcriptional repressor, TOC1, one of five pseudo-response regulators (PRR) that mediate the period of the circadian clock in plants. The TOC1 protein is involved in the clock’s evening loop, which is a repressilator that directly inhibits transcription of morning loop genes LHY and CCA1. Toc1 gene is expressed in most plant structures and cells, and has its locus on chromosome 5.
The TOC1 gene was initially discovered by Prof. Andrew Millar and colleagues in 1995 while Millar was a graduate student. Millar developed an innovative forward genetic screen in which he linked a bioluminescent reporter, firefly (luciferase), to expression of CAB (chlorophyll-a,b binding protein—see Light-harvesting complexes of green plants) in Arabidopsis. By measuring bioluminescence over the course of the day, Millar found CAB expression to display oscillatory patterns in constant light and to oscillate with a shorter period in toc1 mutant plants. He also mapped the toc1 gene to chromosome 5. These methods and discoveries were published in and featured on the cover of Science magazine in February 1995.
Partially because the initial studies of clock genes were conducted in Drosophila in the 1970s and then in mammals, it was originally thought that the plant circadian clock functioned similarly to the mammalian clock. In mammals, positive and negative regulatory elements act in feedback loops to drive circadian oscillations; namely, Per and Cry genes are activated by positive elements CLOCK and BMAL to produce proteins that, when phosphorylated, act as negative elements to inhibit the CLOCK:BMAL complex from its activating function. In this way, Per and Cry inhibit their own transcription.
In contrast, Millar’s group found the TOC1 protein to be a negative regulator, and the plant clock to be better modeled as a repressilator—a system in which one gene represses another and is in turn repressed by the next, forming an interdependent, oscillating gene network. This finding was achieved through 1) Arabidopsis mutants with constitutive (always turned on) toc1 gene expression, which showed decreased mRNA abundance in both morning loop genes prr7 and 9, cca1, and lhy and evening loop genes gi and elf4; and 2) plants with mutations in toc1 and plants in which RNAi was used to knock out toc1. These mutants with no functional toc1 showed an advanced phase for lhy, suggesting less repression in the absence of TOC1 protein.
A study by Carl Strayer and colleagues found that toc1 gene’s transcriptional involvement shortened circadian rhythms in constant dark in addition to constant light, and that TOC1 was circadianly regulated and involved in regulation of its own feedback loop.