UC Davis

Eukaryotic circadian clocks, or oscillators, are composed of transcriptional-translational feedback loops that generate biological rhythms with an approximate twenty-four hour period. These circadian rhythms continue in the absence of environmental cues, which helps organisms adapt to their environment. A family of Myb-like transcription factors act as components of the circadian oscillator in Arabidopsis thaliana and includes a group of repressors and a group of activators that act antagonistically to each other. CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) repress afternoon and evening-phased genes within the oscillator, while REVEILLE 4 (RVE4), REVEILLE 6 (RVE6), and REVEILLE 8 (RVE8) activate these same targets. In this study, I generated a cca1 lhy rve468 quintuple mutant and assessed its circadian and growth phenotypes. Both cca1 lhy and cca1 lhy rve468 mutants have poor circadian rhythms in a range of light conditions and low-amplitude rhythms of core clock gene expression. The cca1 lhy rve468 mutant also flowers early, like cca1 lhy, which suggests that CCA1 and LHY interact epistatically with RVE4, RVE6, and RVE8 to regulate circadian phenotypes. However, hypocotyl elongation and leaf growth in cca1 lhy rve468 mutants are similar to wild type, suggesting that CCA1 and LHY interact additively with RVE4, RVE6, and RVE8 to regulate growth phenotypes. These Myb-like factors therefore have separable functions in circadian regulation and growth. I also generated RVE4, RVE6, and RVE8 single, double, and triple mutants using CRISPR-Cas9 and characterized their growth and circadian phenotypes. My results suggest that the RVEs synergistically regulate flowering time, redundantly regulate leaf growth, and antagonistically regulate hypocotyl elongation. Using these rve mutants, I then investigated light quality-specific circadian regulation of the RVEs and found that blue light-specific enhancement of RVE target gene expression is reduced in rve468 mutants. Additionally, the circadian period of rve468 and rve48 mutants lengthens as fluence rate increases specifically in blue but not red light conditions, while the period of wild type shortens as fluence rate increases in all light qualities. RVE protein abundance and degradation rate are similar in monochromatic red and blue light and are therefore not responsible for the blue light-specific phenotypes of rve mutants. Furthermore, ZEITLUPE (ZTL) and ELONGATED HYPOCOTYL 5 (HY5), which have blue light-specific circadian functions, interact additively with the RVEs to regulate circadian phenotypes. This suggests that the RVEs are involved in light quality-specific circadian regulation through a novel mechanism.