Significance StatementCircadian gene expression oscillates over a 24 hr. period and regulates many genes critical for growth and development. In plants, the Evening Complex (EC), a three-protein repressive complex made up of LUX ARRYTHMO, EARLY FLOWERING 3 and EARLY FLOWERING 4, acts as a key component of the circadian clock and is a regulator of thermomorphogenic growth. However, the molecular mechanisms of complex formation and DNA-binding have not been identified. Here we determine the roles of each protein in the complex and present the structure of the LUX DNA-binding domain in complex with DNA. Based on these data, we used structure-based protein engineering to produce a version of the EC with altered in vitro and in vivo activity. These results demonstrate that the EC can be modified to alter plant growth and development at different temperatures in a predictable manner.
AbstractThe Evening Complex (EC), composed of the DNA-binding protein LUX ARRHYTHMO (LUX) and two additional proteins, EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment and regulates important clock output genes such as PHYTOCHROME INTERACTING FACTOR 4 (PIF4), a key transcription factor involved in temperature dependent plant growth. These properties make the EC an attractive target for altering plant development through targeted mutations to the complex. However, the molecular basis for EC function was not known. Here we show that binding of the EC requires all three proteins and that ELF3 decreases the ability of LUX to bind DNA whereas the presence of ELF4 restores interaction with DNA. To be able to manipulate this complex, we solved the structure of the DNA-binding domain of LUX bound to DNA. Using structurebased design, a LUX variant was constructed that showed decreased in vitro binding affinity but retained specificity for its cognate sequences. This designed LUX allele modulates hypocotyl elongation and flowering. These results demonstrate that modifying the DNAbinding affinity of LUX can be used to titrate the repressive activity of the entire EC, tuning growth and development in a predictable manner. feedback loops repress each other's expression resulting in rhythmic gene expression over a 24-hr period (for review see,(3, 5-7)). The Evening Complex, composed of LUX, ELF3 and ELF4, is a core component of the circadian clock (8-12). The expression patterns of the three genes overlap, peaking at dusk. Thus, the EC has maximum activity at the end of the day and early night, acting to repress expression of the circadian morning loop genes PSEUDORESPONSE REGULATOR 7 and 9 (PRR7 and 9), the central loop gene CCA1 and the evening loop genes GIGANTEA (GI) and LUX itself (12-15).Loss-of-function mutations in elf3, elf4 or lux give rise to arrhythmic circadian outputs with alterations in many developmental pathways (9,(16)(17)(18). This re...