Light quality-dependent roles of REVEILLE proteins in the circadian system.

Several closely related Myb-like activator proteins are known to have partially redundant functions within the plant circadian clock, but their specific roles are not well understood. To clarify the function of the REVEILLE 4, REVEILLE 6, and REVEILLE 8 transcriptional activators, we characterized the growth and clock phenotypes of CRISPR-Cas9-generated single, double, and triple rve mutants. We found that these genes act synergistically to regulate flowering time, redundantly to regulate leaf growth, and antagonistically to regulate hypocotyl elongation. We previously reported that increasing intensities of monochromatic blue and red light have opposite effects on the period of triple rve468 mutants. Here, we further examined light quality-specific phenotypes of rve mutants and report that rve468 mutants lack the blue light-specific increase in expression of some circadian clock genes observed in wild type. To investigate the basis of these blue light-specific circadian phenotypes, we examined RVE protein abundances and degradation rates in blue and red light and found no significant differences between these conditions. We next examined genetic interactions between RVE genes and ZEITLUPE and ELONGATED HYPOCOTYL5, two factors with blue light-specific functions in the clock. We found that the RVEs interact additively with both ZEITLUPE and ELONGATED HYPOCOTYL5 to regulate circadian period, which suggests that neither of these factors are required for the blue light-specific differences that we observed. Overall, our results suggest that the RVEs have separable functions in plant growth and circadian regulation and that they are involved in blue light-specific circadian signaling via a novel mechanism.

Myb-like transcription factors have epistatic effects on circadian clock function but additive effects on plant growth.

The functions of closely related Myb-like repressor and Myb-like activator proteins within the plant circadian oscillator have been well-studied as separate groups, but the genetic interactions between them are less clear. We hypothesized that these repressors and activators would interact additively to regulate both circadian and growth phenotypes. We used CRISPR-Cas9 to generate new mutant alleles and performed physiological and molecular characterization of plant mutants for five of these core Myb-like clock factors compared with a repressor mutant and an activator mutant. We first examined circadian clock function in plants likely null for both the repressor proteins, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), and the activator proteins, REVEILLE 4 (RVE4), REVEILLE (RVE6), and REVEILLE (RVE8). The rve468 triple mutant has a long period and flowers late, while cca1 lhy rve468 quintuple mutants, similarly to cca1 lhy mutants, have poor circadian rhythms and flower early. This suggests that CCA1 and LHY are epistatic to RVE4, RVE6, and RVE8 for circadian clock and flowering time function. We next examined hypocotyl elongation and rosette leaf size in these mutants. The cca1 lhy rve468 mutants have growth phenotypes intermediate between cca1 lhy and rve468 mutants, suggesting that CCA1, LHY, RVE4, RVE6, and RVE8 interact additively to regulate growth. Together, our data suggest that these five Myb-like factors interact differently in regulation of the circadian clock versus growth. More generally, the near-norm al seedling phenotypes observed in the largely arrhythmic quintuple mutant demonstrate that circadian-regulated output processes, like control of hypocotyl elongation, do not always depend upon rhythmic oscillator function.